Certain 7-acylamido-3-(2-carboxyalkyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylmethyl)-3-cephem-4-carboxylic acids their salts and easily hydrolyzed esters

ABSTRACT

Certain 7-acylamido-3-(2-carboxyalkyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acids and their salts and easily hydrolyzed esters of the 4-carboxyl group were synthesized and found to be potent antibacterial agents which exhibited good aqueous solubility. A preferred embodiment was 7-[o-(methylaminomethyl)phenylacetamido]-3-(2-carboxyalkyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of our prior, copendingapplication Ser. No. 701,443 filed June 30, 1976, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The cephalosporins of the present invention in general possess the usualattributes of such compounds and are particularly useful in thetreatment of bacterial infections.

2. Description of the Prior Art

The cephalosporins are a well-known group of semisynthetic antibacterialagents made originally, for example, by acylation of the 7-amino groupof the nucleus 7-aminocephalosporanic acid (7-ACA) and later by similaracylation of nuclei derived therefrom, as by modification of itssubstituent at the 3-position. Various reviews have appeared in thescientific literature (e.g. Cephalosporins and Penicillins -- Chemistryand Biology, edited by Edwin H. Flynn, Academic Press, New York, 1972,and particularly pages 554-569) and in the patent literature, e.g. as inU.S. Pat. Nos. 3,687,948; 3,741,965; 3,743,644; 3,759,904; 3,759,905;3,766,175; 3,766,906; 3,769,281; 3,796,801; 3,799,923; 3,812,116;3,813,388; 3,814,754 and 3,814,755 (all U.S. Class 260-243C).

Issued patents on 3-thiolated cephalosporins in which the 7-substituentis

(A) α-Amino-α-phenylacetamido include U.S. Pat. Nos. 3,641,021,3,734,907, 3,687,948, 3,741,965, 3,757,015, 3,743,644, Japan, No.71/24400 (Farmdoc 46374S), Belgium, No. 776,222 (Farmdoc 38983T; U.K.Pat. No. 1,328,340 which includes various substituents on the benzenering), Belgium Pat. No. 772,592 (Farmdoc 19696T; U.S. Pat. No.3,687,948, 3,734,907 and 3,757,012), West Germany No. 2,202,274 (Farmdoc50428T) corresponding to U.S. Pat. No. 3,759,904, Netherlands, No.7205644 (Farmdoc 76309T; U.S. Pat. No. 3,757,014); and

(B) O-, M- OR P-AMINOETHOXYPHENYLACETAMIDO AS Netherlands, No. 72/13968(Farmdoc 24740U) corresponding to U.S. Pat. No. 3,759,905 and

(C) O-AMINOMETHYLPHENYLACETAMIDO AS U.S. Pat. Nos. 3,766,176 and3,766,175 (which also review the older patent literature concerningsubstituted 7-phenylacetamidocephalosporanic acids) and

(d) N-(phenylacetimidoyl)aminoacetamido as U.S. Pat. No. 3,692,779; and

(e) α-amino-α-(1,4-cyclohexadienyl)acetamido as in Belgium Pat. No.776,222 (Farmdoc 38983T; U.K. Pat. No. 1,328,340).

Additional similar disclosures are found in U.S. Pat. No. 3,692,779(Belgium Pat. No. 771,189; Farmdoc 12819T), Japan, No. 72/05550 (Farmdoc12921T), 72/05551 (Farmdoc 12922T), U.S. Pat. No. 3,719,673 (BelgiumPat. No. 759,570; Farmdoc 39819S), Belgium Pat. Nos. 793,311 (Farmdoc39702U) and 793,191 (Farmdoc 39684U).

Issued disclosures of 3-thiolated cephalosporins in which the7-substituent is 7-mandelamido (7-α-hydroxyphenylacetamido) are found,for example, in U.S. Pat. No. 3,641,021, France 73.10112, U.S. Pat. No.3,796,801, Great Britain Pat. No. 1,328,340 (Farmdoc 38983T), U.S. Pat.No. 3,701,775, Japan 48-44293 (Farmdoc 55334U) and in Hoover et al., J.Med. Chem. 17(1), 34-41 (1974) and Wick et al., Antimicrobial Ag.Chemo., 1(3), 221-234 (1972).

U.S. Pat. No. 3,819,623 (and, for example, also U.K. Pat. No. 1,295,841and West Germany Pat. No. 1,953,861) discloses specifically and withworking details the preparation of 2-mercapto-1,3,4-thiadiazole-5-aceticacid and its conversion to7-(1H-tetrazol-1-yl-acetamido)-3-(5-carboxymethyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid which is also disclosed in West Germany Offenlegungsschrift No.2,262,262.

U.S. Pat. Nos. 3,766,175 and 3,898,217 disclose ##STR1## and ##STR2##respectively.

U.S. Pat. Nos. 3,883,520 and 3,931,160 and Farmdoc Abstract 22850W makereference to 3-heterocyclicthiomethyl cephalosporins containing a numberof substituents (including carboxyl) on the numerous heterocyclesincluded but these references are completely general in nature andinclude no physical constants, yields, methods of synthesis or the likeand do not even name any compound containing a carboxyl substituent.

U.S. Pat. No. 3,928,336 provides a review of much of the oldercephalosporin art.

U.S. Pat. Nos. 3,907,786 and 3,946,000 disclose cephalosporinscontaining various fused ring bicyclic thiols.

Farmdoc abstract 18830X discloses compounds of the formula ##STR3##(where R¹ = acyl or H; R³ = H or methoxy; n = 1-9).

SUMMARY OF THE INVENTION

The present invention provides compounds having the structure: ##STR4##(often written herein as ##STR5## wherein n is one or two and R¹ is acylor hydrogen and R² is hydrogen or the group having the formula ##STR6##wherein, when W represents hydrogen, Z represents (lower)alkanoyl,benzoyl, naphthoyl, furoyl, thenoyl, nitrobenzoyl, methylbenzoyl,halobenzoyl, phenylbenzoyl, N-phthalimido, N-succinimido, N-saccharino,N-(lower)alkylcarbamoyl, (lower)alkoxy, (lower)alkylthio, phenoxy,carbalkoxy, carbobenzoxy, carbamoyl, benzyloxy, chlorobenzyloxy,carbophenoxy, carto-tert.-butoxy or (lower)alkylsulfonyl, and when Wrepresents carbalkoxy, Z represents carbalkoxy and, when W representsphenyl, Z represents benozyl or cyano or wherein W and Z taken togetherrepresent 2-oxocycloalkyl containing 4 to 8 carbon atoms inclusive. Inthe preferred embodiments of this invention R² is hydrogen,pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl,p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl.

As set forth below in more detail the present invention also providessalts of these acids. The stereochemistry of the bicyclic nucleus isthat found in Caphalosporin C.

Acyl (R¹) comprises the groups having the structures: ##STR7## wherein Ris hydrogen, hydroxy or methoxy and R' is hydrogen or methyl.

A preferred embodiment of the present invention consists of thecompounds of Formula I wherein R¹ has the structure ##STR8##

Another preferred embodiment of the present invention consists of thecompounds of Formula I wherein R¹ has the structure ##STR9##

The present invention also provides the process for the production ofthe antibacterial agents having the structure ##STR10## wherein n is oneor two and R¹ is acyl as defined above which comprises reacting acompound of the formula ##STR11## wherein n is one or two or a salt oreasily hydrolyzed ester or Schiff base as with benzaldehyde orsalicylaldehyde thereof (including, but not limited to, those of U.S.Pat. No. 3,284,451 and U.K. Pat. No. 1,229,453 and any of the silylesters described in U.S. Pat. No. 3,249,622 for use with6-aminopenicillanic acid and used in Great Britain Pat. No. 1,073,530and particularly the pivaloyloxymethyl, acetoxymethyl, methoxymethyl,acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyland 5-indanyl esters) thereof with an organic monocarboxylic acidchloride or a functional equivalent thereof as an acylating agent.

Such functional equivalents include the corresponding acid anhydrides,including mixed anhydrides and particularly the mixed anhydridesprepared from stronger acids such as the lower aliphatic monoesters ofcarbonic acid, or alkyl and aryl sulfonic acids and of more hinderedacids such as diphenylacetic acid. In addition, an acid azide or anactive ester or thioester (e.g. with p-nitrophenyl, 2,4-dinitrophenol,thiophenol, thioacetic acid) may be used or the free acid itself may becoupled with compound II after first reacting said free acid withN,N'-dimethylchloroformiminium chloride [cf. Great Britain Pat. No.1,008,170 and Novak and Weichet, Experientia XXI, 6, 360 (1965)] or bythe use of enzymes or of an N,N'-carbonyldiimidazole or anN,N'-carbonylditriazole [cf. South African patent specification 63/2684]or a carbodiimide reagent [especially N,N'-dicyclohexylcarbodiimide.N,N'-diisopropylcarbodiimide orN-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide; cf. Sheehan and Hess,J. Amer. Chem. Soc. 77, 1967 (1955)], or of alkylamine reagent [cf. R.Buijle and H. G. Viehe, Angew. Chem. International Edition 3, 582,(1964)] or of an isoxazolium salt reagent [cf. R. B. Woodward, R. A.Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)], or of aketenimine reagent [cf. C. L. Stevens and M. E. Munk, J. Amer. Chem.Soc., 80, 4065 (1958)] or of hexachlorocyclotriphosphatriazine orhexabromocyclotriphosphatriazine (U.S. 3,651,050) or ofdiphenylphosphoryl azide [DPPA; J. Amer. Chem. Soc., 94, 6203-6205(1972)] or of diethylphosphoryl cyanide [DEPC; Tetrahedron Letters No.18, pp. 1595-1598 (1973)] or of diphenyl phosphite [Tetrahedron LettersNo. 49, pp. 5047-5050 (1972)]. Another equivalent of the acid chlorideis a corresponding azolide, i.e., an amide of the corresponding acidwhose amide nitrogen is a member of a quasiaromatic five membered ringcontaining at least two nitrogen atoms, i.e., imidazole, pyrazole, thetriazoles, benzimidazole, benzotriazole and their substitutedderivatives. As an example of the general method for the preparation ofan azolide, N,N'-carbonyldiimidazole is reacted with a carboxylic acidin equimolar proportions at room temperature in tetrahydrofuran,chloroform, dimethylformamide or a similar inert solvent to form thecarboxylic acid imidazolide in practically quantitative yield withliberation of carbon dioxide and one mole of imidazole. Dicarboxylicacids yield diimidazolide. The by-product, imidazole, precipitates andmay be separated and the imidazolide isolated, but this is notessential. The methods for carrying out these reactions to produce acephalosporin and the methods used to isolate the cephalosporin soproduced are well known in the art.

Mention was made above of the use of enzymes to couple the free acidwith compound II. Included in the scope of such processes are the use ofan ester, e.g. the methyl ester, of that free acid with enzymes providedby various microorganisms, e.g. those described by T. Takahashi et al.,J. Amer. Chem. Soc., 94(11), 4035-4037 (1972) and by T. Nara et al., J.Antibiotics (Japan) 24(5), 321-323 (1971) and in U.S. Pat. No.3,682,777.

For the coupling of the organic carboxylic acid as described above withcompound II (or a salt or preferably an easily hydrolyzed ester ofSchiff base, as with benzaldehyde, thereof) it is also convenient andefficient to utilize as the coupling agent phosphonitrilic chloridetrimer (J. Org. Chem., 33(7), 2979-81, 1968) orN-ethoxy-1,2-dihydroquinoline (EEDQ) as described in J. Amer. Chem.Soc., 90, 823-824 and 1652-1653 (1968) and U.S. Pat. No. 2,455,929. Thereaction is preferably carried out at 30°-35° C. in benzene, ethanol ortetrahydrofuran using about equimolar quantities of all three reagentsfollowed by conventional isolation and removal by conventional methodsof any blocking groups present.

An additional process of the present invention comprises the preparationof the compounds of the present invention by the displacement of the3-acetoxy group of a 7-acylaminocephalosporanic acid (prepared bysubstituting 7-aminocephalosporanic acid for the3-thiolated-7-aminocephalosporanic acids in the acylation proceduresdescribed herein and elsewhere reported) with a thiol HSR³ having theformula ##STR12## wherein n is one or two and then removing theprotecting group if any is present, as on the aminomethyl ormethylaminomethyl group or on the carboxyl group or both. Thedisplacement of such a 3-acetoxy group with such a thiol may beaccomplished in solution as in water or aqueous acetone at a temperatureof at least room temperature and preferably within the range of about50° to 100° C. in the presence of a mild base such as sodiumbicarbonate, e.g. preferably near neutrality such as at about pH 6. Anexcess of the thiol is preferably employed. The reaction product isisolated by careful acidification of the reaction mixture followed byextraction with a water-immiscible organic solvent. As noted above, thepreparation of many other 7-acylamidocephalosporanic acids is describedin the patent and scientific literature, e.g. in U.S. Class 260-243C.

When the organic carboxylic acid contains a functional group such asamino or methylamine it is often desirable to first block (or protect)said group, then carry out the coupling reaction and finally subject theresulting compound to chemical removal of the protecting group, that is,subjecting the resulting compound to elimination reaction of theprotecting group.

The salts of the compounds of this invention include the nontoxiccarboxylic acid salts thereof, including nontoxic metallic salts such assodium, potassium, calcium and aluminum, the ammonium salt andsubstituted ammonium salts, e.g. salts of such nontoxic amines astrialkylamines including triethylamine, procaine, dibenzylamine,N-benzyl-beta-phenethylamine, 1-ephenamine,N,N'-dibenzylethylenediamine, dehydroabietylamine,N,N'-bis-dehydroabietylethylenediamine, N-(lower)alkylpiperidine, e.g.N-ethylpiperidine, and other amines which have been used to form saltswith benzylpenicillin; and the nontoxic acid addition salts thereof(i.e., the amine salts) including the mineral acid addition salts suchas the hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate andphosphate and the organic acid addition salts such as the maleate,acetate, citrate, oxalate, succinate, benzoate, tartrate, fumarate,malate, mandelate, ascorbate and the like.

Also included in this invention are the compounds (used as eitherintermediates or metabolic precursors) in which the amino group is"blocked" by substituents such as 2-iodoethoxycarbonyl (U.K. Pat. No.1,349,673), t-butoxycarbonyl, carbobenzyloxy, formyl,o-nitrophenylsulfenyl, β,β,β-trichloroethoxycarbonyl,4-oxo-2-pentenyl-2, 1-carbomethoxy-1-propenyl-2- and the like.Particularly included in such blocking groups are the ketones(especially acetone) and aldehydes (especially formaldehyde andacetaldehyde) disclosed, for example, in U.S. Pat. Nos. 3,198,804 and3,347,851 and the β-ketoesters and β-diketones disclosed, for example,in U.S. Pat. No. 3,325,479 and the β-ketoamides disclosed in Japan No.71/24714 (Farmdoc 47, 321S).

The preferred esters of the cephalosporins of the present invention arethe pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl andphenacyl esters. All are useful intermediates in the production of thecephalosporin having a free carboxyl group.

As indicated above, these five esters of 7-aminocephalosporanic acid areeach prepared by known methods. One excellent procedure is that of U.S.Pat. No. 3,284,451 in which sodium cephalothin is esterified by reactionwith the corresponding active chloro or bromo compound (e.g. phenacylbromide, chloroacetone, chloromethyl ether, pivaloyloxymethyl chloride[also called chloromethyl pivalate], acetoxymethyl chloride) and thenthe thienylacetic acid sidechain is removed enzymatically as in the samepatent or chemically as in U.S. Pat. No. 3,575,970 and in Journal ofAntibiotics, XXIV (11), 767-773 (1971). In another good method thetriethylamine salt of 7-aminocephalosporanic acid is reacted directlywith the active halogen compound, as in United Kingdom Pat. No.1,229,453.

These esters of 7-aminocephalosporanic acid are then reacted with thenucleophile HSR³ in the same manner as is illustrated herein for7-aminocephalosporanic acid itself. The 3-thiolated ester of7-aminocephalosporanic acid is then coupled with the organic carboxylicacid R'--OH as before. Before or after removal of any blocking group,e.g. on an amino group in the 7-sidechain, the ester of thecephalosporin so obtained is, if not used per se, converted to its freeacid, including its zwitterion (and, if desired, any salt) by removal ofthe esterifying group, as by aqueous or enzymatic hydrolysis (as withhuman or animal serum) or acidic or alkaline hydrolysis or by treatmentwith sodium thiophenoxide as taught in U.S. Pat. No. 3,284,451 and, inthe penicillin series, by Sheehan et al., J. Org. Chem. 29(7), 2006-2008(1964).

In another alternative synthesis, the 3-thiolated 7-aminocephalosporanicacid is prepared as described herein and then acylated at the 7-aminogroup and finally esterified, as by reaction of the appropriate alcoholwith the acid chloride prepared, for example, by reaction of the finalcephalosporin with thionyl chloride or by other essentially acidicesterification procedures.

In the treatment of bacterial infections in man, the compounds of thisinvention are administered parenterally in an amount of from about 5 to200 mg./kg./day and preferably about 5 to 20 mg./kg./day in divideddosage, e.g. three to four times a day. They are administered in dosageunits containing, for example, 125, 250 or 500 mg. of active ingredientwith suitable physiologically acceptable carriers or excepients. Thedosage units are in the form of liquid preparations such as solutions orsuspensions.

The other reagents used to prepare the compounds of the presentinvention are synthesized either as described in the art (e.g. as in thepatents and publications noted above) or by strictly analogousprocedures. For convenience and purposes of illustration, however, thereare given below some specific examples of such syntheses, e.g. toprepare carboxylic acids containing a free amino group which is"blocked" with tert.-butoxycarbonyl.

2-(tert.-Butoxycarbonylaminomethyl)-1,4-cyclohexadienylacetic acid

A solution of 16.5 g. (0.1 mole) of o-aminomethylphenylacetic acid in1.5 l of liquid ammonia (which had been treated with 50 mg. of Li toremove a trace of moisture) was slowly diluted with 500 ml. of dryt-BuOH. To the solution was added in small portions 3.4 g. (0.5 atom) ofLi over a period of 4 hours and the mixture was stirred for 16 hours atroom temperature removing the liquid ammonia in a hood and finallyevaporated to dryness below 40° C. The residue was dissolved in 500 ml.of water and the solution was chromatographed on a column of IR-120 (H⁺,700 ml.) resin and eluted with 1% NH₄ OH solution. Ninhydrin positivefractions of the eluate were combined and evaporated to dryness. Theresidue was washed with four 50 ml. portions of hot acetone andrecrystallized from 500 ml. of ethanol-water (1:1) to give 11.2 g. (67%)of colorless needles, o-( 2-aminomethyl)-1,4-cyclohexadienyl)aceticacid. M.p. 183° C.

IR: ν_(max) ^(nuj) 1630, 1520, 1380, 1356 cm⁻¹.

NMR: δ^(D).sbsp.2^(O) + K.sbsp.2^(CO).sbsp.3 ##STR13## 3.01 (2H, s, CH₂CO), 3.20 (2H, s, CH₂ --N), ##STR14##

Anal. Calcd. for C₉ H₁₃ NO₂ : C, 64.65; H, 7.84; N, 8.38. Found: C,64.77; H, 8.06; N, 8.44.

Improved Procedure for the Preparation ofα-(2-aminomethyl-1,4-cyclohexadienyl)-acetic acid ##STR15##

The procedure used by Welch, Dolfini and Giarrusso in U.S. Pat. No.3,720,665 (Example 1) to make D-2-amino-2-(1,4-cyclonexadienyl)aceticacid was adapted. A solution of 830 ml. of distilled liquid ammonia wasdried with 40 mg. of lithium under an argon atmosphere. To this stirredsolution was added 11.0 g. (0.07 mole) of 2-aminomethylphenylacetic acidand 340 ml. of tert. butyl alcohol. A total of 1.6 g. (0.225 mole) oflithium was added to the vigorously stirred solution over a period of 2hours. The grey mixture was then treated with 35 g. (0.215 mole) oftriethylamine (TEA) hydrochloride and stirred overnight at roomtemperature for 18 hours. The tert. butyl alcohol was removed at 40° (15mm.) to yield a white residue which was dried in vacuo over P₂ O₅overnight. The solid was dissolved in 30 ml. of 1:1 methanol-water andadded with stirring to 3.5 l. of 1:1 chloroform-acetone at 5°. Themixture was stirred for 20 min. and the amino acid,α-(2-aminomethyl-1,4-cyclohexadienyl)acetic acid, was collected anddried for 16 hours in vacuo over P₂ O₅ to yield 6.3 g. (58%) of whitecrystals, m.p. 190° decomp. The IR and NMR spectra were consistent forthe structure.

A solution of 19.31 g. (0.135 m) of tert.-butoxycarbonylazide in 152 ml.of tetrahydrofuran (THF) was added to a stirred solution of 14.89 g.(0.09 m) of 2-aminomethyl-1,4-cyclohexadienylacetic acid and 7.20 g.(0.18 m) of sodium hydroxide in 281 ml. of water. The solution wasstirred for 18 hr. at 25° and then filtered thru diatomaceous earth(Super-cel). The THF was removed at 40° (15 mm) and the residualsolution was washed with ether (2 × 175 ml.) and acidified with 6 Nhydrochloric acid (HCl). The mixture was stirred in an ice-bath and theprecipitate was collected and dried for 18 hr. in vacuo over P₂ O₅ at25° to yield 17.3 g. (72.6%) of2-(tert.-butoxycarbonylaminomethyl)-1,4-cyclohexadienylacetic acid as awhite powder. The IR and NMR spectra were consistent for the structure.

Preparation of 3-Aminomethyl-2-thiophene Acetic Acid ##STR16## A.Thiophene-3-carboxaldehyde Dimethyl Acetal (2a)

A mixture of thiophene-3-carboxaldehyde¹ (322 g., 2.9 moles),trimethoxymethane (636 g., 6 moles) and IR-120 resin (H⁺, 6 g.) inmethanol (200 ml.) was refluxed over a period of 4 hours. The resin wasremoved and the filtrate was evaporated under reduced pressure to give acolorless oil which was distilled under reduced pressure. Yield 423 g.(94%), b.p. 90°-95° C. 13 mmHg.

ir: ν_(max) ^(liq) 3150, 1045, 1025 cm⁻¹

nmr: δ_(ppm) ^(neat) 3.21 (6H, s, OCH₃), ##STR17## 7.0-7.4 (3H, m,thiophene-H)

B. 2-Formylthiophene-3-carboxaldehyde Dimethylacetal (3a)

To a stirred solution of 2a (423 g., 2.68 moles) in anhydrous ether (1L) was added dropwise in 1 hour a freshly prepared solution ofn-butyllithium (27 moles) in ether keeping a gentle reflux under dry N₂.Reflux being continued for 0.5 hour, a solution of DMF(dimethylformamide) (432 g., 6 moles) in anhydrous ether (0.8 L) wasadded dropwise to the mixture over a period of 0.75 hour with vigorousstirring. After the complete addition the mixture was stirred overnight,poured onto crushed ice (1 Kg.) with stirring and allowed to rise toroom temperature. The organic layer was separated and the water layerwas saturated with NaCl and extracted thoroughly with ether (2 × 200ml.). The ether extracts were combined, dried over MgSO₄ andconcentrated. The residue was distilled under reduced pressure and thepale yellow oil was collected at 100°-125° C., 0.7 mmHg. Yield, 277 g.(56%).

ir: ν_(max) ^(liq) 3110, 1660, 1100 cm⁻¹.

nmr: δ_(ppm) ^(neat) 3.40 (6H, s, OCH₃), ##STR18## 7.27 (1H, d, J=6Hz,thiophene-Hβ), 7.81 (1 H, d-d, J=1.5 and 6Hz, thiophene-Hα), 10.34 (1H,d, J=1.5 Hz, --CHO).

C.1-Methylsulfinyl-1-methylthio-2-(3-carboxaldehyde-ethyleneacetal-2-thienyl)ethylene(4b)

Preparation of 4b was carried out according to the procedure similar tothat reported by K. Ogura et al.⁴ Triton B (40% in methanol, 2 ml. inTHF (tetrahydrofuran) (5 ml.) was added to a solution of methylmethylthiomethyl sulfoxide² (2.5 g., 20 m. moles) and2-formyl-3-thiophenecarboxaldehyde ethylene acetal³ (3b). The mixturewas refluxed for about one hour and concentrated under reduced pressure.The residue was dissolved in benzene (150 ml) and extracted with water(3 × 20 ml). The organic layer was dried over MgSO₄ and evaporated todryness under reduced pressure. The residue was column-chromatographedon silica gel (80 g) eluting with benzene (500 ml) and chloroform (500ml) successively. From the chloroform eluate 4.9 g (85%) of the product4b was isolated as a pale yellow oil.

ir: ν_(max) ^(liq) 3110, 1600 cm⁻¹.

nmr: δ_(ppm) ^(CDCl).sbsp.3 2.42 (3H, s, S--CH₃), 2.78 (3H, s, SO--CH₃),4.15 (4H, m, CH₂ CH₂ --), ##STR19## 7.34 (1H, d, J=4.5Hz, thiophene-Hβ),7.40 (1H, d, J=4.5Hz, thiophene-Hα), 8.28 (1H, s, --CH═).

The semicarbazone of 4 was prepared by a usual manner and crystallizedfrom ethanol-DMF. M.p. 212°-213° C.

Anal. Calcd. for C₁₀ H₁₃ N₃ O₂ S₂ : C, 39.58; H, 4.32; N, 13.85; S,31.70. Found: C, 39.46; H, 4.24; N, 14.05; S, 31.63.

D. 1-Methylsulfinyl-1-methylthio-2-(3-carboxaldehydedimethylacetal-2-thienyl)ethylene (4a)

The compound 4a was prepared by the procedure similar to that for 4b.Triton B (40% in methanol, 50 ml) was added to a solution of methylmethylthiomethylsulfoxide (72 g., 0.58 mole) and 3a (108 g, 0.58 mole)in THF (300 ml) and the mixture was refluxed for 4 hours. Separation bycolumn chromatography with silica gel (400 g) eluting with chloroform (5L) gave 130.5 g (78%) of 4a as a pale yellow oil.

ir: ν_(max) ^(liq) 3100, 1580, 1100, 1050⁻¹.

nmr: δ_(ppm) ^(CCl).sbsp.4 2.42 (3H, s, S--CH₃), 2.70 (3H, s, SO--CH₃),3.34 (6H, s, OCH₃), ##STR20## 7.20 (1H, d, J=6Hz, thiophene-Hβ), 7.40(1H, d, J=6Hz, thiophene-Hα), 8.12 (1H, s, --CH═).

E. Ethyl 3-formyl-2-thienylacetate⁴ (5)

Dry hydrogen chloride (33 g) was absorbed in anhydrous ethanol (500 ml).To this solution 4a (130 g, 0.45 mole) was added and the mixture heatedunder reflux for 5 mins. The reaction mixture was diluted with water andevaporated under reduced pressure. The residue was extracted withbenzene (2 × 100 ml) and the benzene extracts were combined, washed withwater (50 ml), dried over MgSO₄ and evaporated to dryness. The oilyresidue was column-chromotographed on silica gel (400 g) eluting withchloroform (5 L). Fractions containing the desired product were combinedand concentrated. The residual oil (60 g) was distilled under reducedpressure to afford 23 g (23%) of 5, boiling at 120°-126° C/l mmHg.

ir: ν_(max) ^(liq) 3110, 1730, 1670 cm⁻¹.

nmr: δ_(ppm) ^(CDCl).sbsp.3 1.30 (3H, t, J=6Hz, --CH₂ CH₃), 4.25 (2H, q,J=6Hz, --CH₂ CH₃), 4.26 (2H, s, --CH₂ CO), 7.25 (1H, d, J=5Hz,thiophene-Hβ), 7.48 (1H, d, J=5Hz, thiophene-Hα), 10.15 (1H, s, CHO).

The analytical sample of 5 was submitted as the2,4-dinitrophenylhydrazone which was crystallized from chloroform. M.p.178°-179° C.

ir: ν_(max) ^(nujol) 1720, 1610, 1570 cm⁻¹.

Anal. Calcd. for C₁₅ H₁₄ N₄ O₆ S: C, 47.62; H, 3.73; N, 14.81; S, 8.47.Found: C, 47.33; H, 3.47; N, 14.77; S, 8.68.

According to the similar procedure 2.2 g (7.6 m moles) of the ethyleneacetal 4b was treated with 1.1 g of dry hydrogen chloride in 800 ml ofanhydrous ethanol to afford 5 which was purified by columnchromatography on silica gel (30 g). Elution with chloroform gave 663 mg(44%) of 5 as a pale yellow oil.

F. Ethyl 3-formyl-2-thienylacetate oxime (6)

Sodium carbonate (1.7 g, 16 m mole) was added to a solution of thealdehyde 5, (3.14 g, 16 m mole) and hydroxylamine hydrochloride (2.2 g,32 m mole) in 50% aq. ethanol (40 ml) at 5° C with stirring. Thereaction mixture was warmed up to room temperature. After 2.5 hrs., thereaction mixture was concentrated under reduced pressure. The residuewas extracted with benzene (3 × 50 ml). The benzene extracts were washedwith water (10 ml), dried over MgSO₄, and evaporated under reducedpressure. Separation by column chromatography on silica gel (60 g) gave2.7 g (80%) of colorless oil 6.

ir: ν_(max) ^(liq) 3400, 1730, 1620 cm⁻¹.

nmr: δ_(ppm) ^(Aceton-d).sbsp.6 1.23 (3H, t, J=7.5Hz, --CH₂ CH₃), 4.01(2H, s, --CH₂ CO), 4.14 (2H, q, J=7.5Hz, --CH₂ CH₃), 7.31 (2H, s,thiophene-H), 8.26 (1H, s, --CH═N), 10.15 (1H, s, NOH, disappeared byaddition of D₂ O).

G. The δ-lactam of 3-aminomethyl-2-thienylacetic acid (7).

Method A: Catalytic reduction

A mixture of the oxime 6 (2.65 g, 12.4 m moles), 10% palladium oncharcoal, dry hydrogen chloride (1.4 g, 37.2 m moles) in anhydrousethanol (68 ml) was hydrogenated overnight under atmospheric pressure atroom temperature. The catalyst was exchanged twice and the reaction wascarried out over a period of 3 days. The catalyst was removed and thefiltrate was concentrated under reduced pressure. To the residue wasadded water (10 ml) and the mixture washed with ethyl acetate (2 × 10ml). The aqueous layer was adjusted to pH 9 with sodium carbonate,saturated with sodium chloride, and extracted with ethyl acetate (3 × 20ml). The ethyl acetate extracts were dried over MgSO₄, treated withcharcoal, and evaporated under reduced pressure. Recrystallization fromethyl acetate gave 417 mg (22%) of colorless needles 7 melting at194°-195° C.

ir: ν_(max) ^(KBr) 3200, 1650, 1480 cm⁻¹.

nmr: δ_(ppm) ^(DMSO-d).sbsp.6 3.53 (2H, t, J=3Hz, --CH₂ CO--), 4.36 (2H,d-t, J=3, 1.5Hz, changed to a triplet by addition of D₂ O, J=3Hz, CH₂N), 6.95 (1H, d, J=4.5Hz, thiophene-Hβ), 7.45 (1H, d, J=4.5Hz,thiophene-Hα), 8.0 (1H, m, disappeared by addition of D₂ O, NH).

Anal. Calcd. for C₇ H₇ NOS: C, 54.88; H, 4.61; N, 9.14; S, 20.93. Found:C, 55.04; H, 4.45; N, 9.13; S, 20.50.

method B: Zn-dust reduction

To a solution of the oxime 6 (18.3 g, 86 m moles) in acetic acid (200ml), zinc dust (17 g, 258 m moles) was added portionwise over a periodof 1 hr. at 40°-50° C with vigorous stirring. The reaction mixture wasstirred overnight at room temperature and heated at 60° C for 4 hours.The contents were filtered and the filtrate was concentrated underreduced pressure. To the residual oil was added water (100 ml) and themixture washed with ether (2 × 50 ml). The aqueous solution was layeredwith ethyl acetate (100 ml) and adjusted to pH 10 with sodium carbonate.The precipitate was filtered off. The filtrate was extracted with ethylacetate. The ethyl acetate extracts were washed with water (10 ml),dried over MgSO₄, and evaporated under reduced pressure. The residualsolid was triturated with benzene. Crystallization from ethyl acetategave 2.7 g (21%) of the lactam 7 which was identical to Method A in theIR and the NMR spectra.

H. 3-Aminomethyl-2-thienylacetic acid (8)

A mixture of the lactam 7 (2.88 g, 18.8 m moles) and 6N hydrochloricacid (50 ml) was heated under reflux for 3 hrs. The reaction mixture wasconcentrated under reduced pressure. To the residue was added water (20ml) and the mixture treated with charcoal and evaporated under reducedpressure. The trituration of the residue with THF gave the amino acid 8hydrochloride (3.72 g, 95%; m.p. 171°-172° C; ir (KBr) cm⁻¹ : 3450,3000, 1700, 1200; nmr (D₂ O)ppm: 4.80 (2H, s, --CH₂ CO), 4.27 (2H, s,CH₂ --N), 7.26 (1H, d, J=6Hz, thiophene-Hβ), 7.53 (1H, d, J=6Hz,thiophene-Hα). The hydrochloride (3.71 g, 17.9 m moles) was dissolved inwater (10 ml) chromatographed on a column of IR-120 (H, 30 ml) anddeveloped successively with water (100 ml) and 5N--NH₄ OH (2 L). Theammonia eluate was evaporated to dryness. The residue was crystallizedfrom aqueous acetone to give 3.0 g (98%) of 8, m.p. 223°-225° C.

ir: ν_(max) ^(KBr) 3000, 1620, 1520 cm⁻¹.

nmr: δ_(ppm) ^(D).sbsp.2^(O--Na).sbsp.2^(CO).sbsp.3 3.20 (2H, s, --CH₂CO), 4.13 (2H, s, CH₂ N), 7.04 (1H, d, J=6Hz, thiophene-Hβ), 7.30 (1H,d, J=6Hz, thiophene-Hα).

Anal. Calcd. for C₇ H₉ NO₂ S: C, 49.10; H, 5.30; N, 8.18; S, 16.73.Found: C, 48.53: h, 5.22; N, 7.98; S, 18.97.

I. 3-t-Butoxycarbonylaminomethyl-2-thienylacetic acid (9)

A mixture of 3-aminomethyl-2-thienylacetic acid 8 (3.1 g, 18 m. moles)and triethylamine (8 g. 80 m moles) in 50% aqueous acetone (80 ml) wasadded dropwise t-butoxycarbonyl azide (5.7 g, 40 m moles) over a periodof 20 mins. at 0° C with vigorous stirring. The reaction mixture wasstirred overnight at room temperature and concentrated under reducedpressure. The concentrate was washed with ether (2 × 20 ml), adjusted topH 2 with conc. HCl and extracted with ethyl acetate (2 × 50 ml). Theethyl acetate extracts were washed with saturated aqueous sodiumchloride, dried over MgSO₄, treated with charcoal and evaporated underreduced pressure. The residue was triturated with n-hexane andcrystallized from n-hexane and benzene to give 4.5 g (92%) of colorlessneedles 9, melting at 62° - 63° C.

ir: ν_(max) ^(nujol) 3350, 1700 cm⁻¹.

nmr: δ_(ppm) ^(CDCl).sbsp.3 1.43 (9H, s, BOC--H), 3.27 (2H, s, CH₂ CO),4.16 (2H, d, J=6Hz, CH₂ -N, a singlet when D₂ O was added), 5.00 (1H,br, --NH--, disappeared by addition of D₂ O), 6.30 (1H, broad s, --COOH,disappeared by addition of D₂ O), 6.86 (1H, d, J=6Hz, thiophene-Hβ),7.06 (1H, d, J=6Hz, thiophene-Hα).

Anal. Calcd. for C₁₂ H₁₇ NO₄ S: C, 52.89; H, 6.29; N, 5.14; S, 11.77.Found: C, 53.30; H, 6.39; N, 5.13; S, 11.72.

Preparation of 2-N-Methylaminomethyl-4-methoxy-(and4-hydroxy-)phenylacetic Acids ##STR21##2-N-Tosylaminomethyl-4-hydroxyphenylacetic Acid (2)

To a solution of 14.56 g. (0.08 mol.) of2-aminomethyl-4-hydroxyphenylacetic acid (1) (U.S. Pat. No. 3,823,141)and 13 g. (0.32 mol.) of sodium hydroxide in 200 ml. of water was addeddropwise with stirring at 65°-70° C. a solution of 18.5 g. (0.097 mol.)of p-toluenesulfonyl chloride in 50 ml. of dry ether and the mixture waskept at the same temperature for one hour. The mixture being cooled, theaqueous layer was separated, washed with ether (2 × 50 ml.), acidifiedwith 6N HCl and extracted with 400 ml. of ethyl acetate. The extract waswashed with water and a saturated aqueous NaCl solution, dried with Na₂SO₄ and treated with active carbon (1 g.). The filtrate was concentratedto dryness and the residue was crystallized from ethyl acetate to give11.0 g. (40.5%) of 2 melting at 212°-215° C.

ir: ν _(max) ^(KBr) 3240, 1700, 1380, 1330, 1150 cm⁻¹

uv: ν _(max) ^(1%K).sbsp.2^(CO).sbsp.3 230 nm (ε: 7,750)

nmr: δ _(ppm) ^(DMSO-d).sbsp.6 2.47 (3H, s, Ar--CH₃), 3.60 (2H, s, CH₂CO), 3.93 (2H, d,J=6.0 Hz, CH₂ N), 6.6-8.2 (7H, m, phenyl-H).

2-(N-Methyl-N-tosylamino)methyl-4-methoxyphenylacetic Acid (3)

A mixturwe of 11 g. (0.033 mol.) of 2, 10.3 ml. (0.17 mol.) of methyliodide and 9.2 g. (0.24 mol.) of sodium hydroxide in 100 ml. of waterwas heated at 80°-90° C. for 45 minutes in a sealed tube with occasionalshaking. The mixture was washed with ethyl acetate (30 ml.) and thewater layer was acidified with 6N HCl and extracted with ethyl acetate(3 × 30 ml.). The combined extracts were washed with water (30 ml.) anda saturated aqueous NaCl solution (30 ml.) treated with active carbon (1g.) and dried over Na₂ SO₄. The filtrate was evaporated to dryness andthe residue was crystallized from benzene to give 8 g. (66.5%) of theN,O-dimethyl derivative 3 melting at 146°-150° C.

ir: ν _(max) ^(KBr) 1690, 1500, 1340, 1280, 1150 cm⁻¹.

uv: λ _(max) ^(EtOH) 229 nm (ε: 20500), 278 nm (ε: 2400).

nmr: δ _(ppm) ^(DMSO-d).sbsp.6 2.52 (3H, s, N--CH₃), 2.47 (3H, s,Ar--CH₃), 3.67 (2H, s, CH₂ CO), 3.74 (3H, s, OCH₃), 4.10 (2H, s, CH₂ N),6.7-7.8 (7H, m, Ar--H), 11.5 (1H, br-s, COOH).

Anal. Calc'd. for C₁₈ H₂₁ NO₅ S: C, 59.49; H, 5.82; N, 3.84; S, 8.82.Found: C, 59,48; H, 5.68; N, 3.37; S, 9.22

2-N-Methylaminomethyl-4-methoxyphenylacetic Acid (4a)

To a solution of liquid ammonia (300 ml.) was added 9.4 g. (0.026 mol.)of 3 at -50° C. and the mixture was stirred until a clear solution wasobtained at the same temperature. To the solution was added 3.3 g. (0.14g. atom) of Na in small pieces at -40° C. and the mixture was stirredfor 2 hours. Ammonia was evaporated amd the residue was dissolved in 100ml. of water carefully. To the solution was added 100 ml. of AmberliteIR-C 50 (ammonium type) and the mixture was stirred for 30 minutes atroom temperature. The resin was removed and the filtrate was treatedwith barium acetate until no more precipitate was observed. Theprecipitate was filtered off and the filtrate was chromatographed with acolumn of IR-120 ion-exchange resin (H⁺, 100 ml.) by eluting with 5-10%ammonia. The eluate (2 L) containing the desired product was evaporatedto dryness below 50° C. and the residue was triturated with acetone togive 4.4 g. (81%) of 4a, m.p. 225°-227° C.

ir: ν _(max) ^(KBr) 1590, 1380, 1260, 1035 cm⁻¹.

nmr: δ _(ppm) ^(D).sbsp.2^(O) 2.77 (3H, s, N--CH₃), 3.6 (2H, s, CH₂ CO),3.87 (3H, s, OCH₃), 4.18 (2H, s, CH₂ N), 6.8-7.4 (3H, m, phenyl-H).

2N-Methylaminomethyl-4-hydroxyphenylacetic Acid (4b)

A mixture of 2.9 g. (0.014 mol.) of 4a in 30 ml. of 48% hydrobriomicacid was refluxed for 5 hours and the solution was evaporated todryness. The residue was dissolved in 50 ml. of water. The solution waschromatographed on a column of Amberlite IR-120 (H⁺, 50 ml.) elutingwith 5-10% ammonia. The eluate was collected in 250 ml. fractions.Fractions containing the product were combined and evaporated to drynessbelow 50° C. The residue was triturated with acetone to give 1.3 g.(48.5%) of 4b, which was crystallized from 80% ethanol. M.p. 218°-221°C.

ir: ν _(max) ^(KBr) 2000-3400, 1610, 1540, 1460, 1380, 1270 cm⁻¹.

uv: λ _(max) ^(1%K).sbsp.2^(CO).sbsp.3 243 mn (ε: 4700), 297 nm (ε:1350).

nmr: δ _(ppm) ^(D).sbsp.2^(O+NaOH) 2.64 (3H, s, N--CH₃), 3.47 (2H, s,CH₂ CO), 3.94 (2H, s, N--CH₂), 6.5--7.2 (3H, m, phenyl-H).

Anal. Calc'd for C₁₀ H₁₃ NO₃ : C, 61.53; H, 6.71; N, 7.17. Found: C,61.44; H, 6.81; N, 7.20.

2N-t-Butoxycarbonyl-N-methylaminomethyl-4-methoxyphenylacetic Acid (5, R= CH₃)

A mixture of 1.05 g. (5 m.mol.) of 4a, 1.43 g. (6 m.mol.) of t-butyl4.6-dimetylpyrimidin-2-ylthiolcarbonate and 1.4 ml. of triethylamine in40 ml. of 50% THF was stirred at room temperature for 20 hours. Most ofthe THF was evaporated and the resulting aqueous solution (ca. 20 ml.)was washed with ether. The water layer was acidified witg 6N HCl andextracted with ether (3 × 10 ml.). The ethereal extracts were washedwith water (10 ml.) and a saturated aqueous NaCl solution (10 ml.),treated with a small amount of active carbon and dried over Na₂ SO₄. Thefiltrate was evaporated to dryness to give 1.0 g. (77.5%) of 5 (R = CH₃)as an oil.

nmr: δ _(ppm) ^(CDCl).sbsp.3 1.47 (9H, s, BOC--H), 2.77 (3H, s, N--CH₃),3.60 (2H, s, CH₂ CO), 3.79 (3H, s, O--CH₃), 4.49 (2H, s, CH₂ N), 6.1-7.3(3H, m, phenyl-H).

2-N-t-Butoxycarbonyl-N-methylaminomethyl-4-hydroxyphenylacetic Acid (5,R =H)

A mixture of 1 g. (4.78 m.mol.) of 4b, 1.5 g. (6.3 m.mol.) of t-butyl4,6-dimethylpyrimidin-2-ylthiolcarbonate and 2.1 ml. of triethylamine in50 ml. of 50% aqueous THF solution was stirred at room temperture for 20hours. The mixture was concentrated to 20 ml. under reduced pressure.The concentrate was washed with ether (10 ml.), acidified with 6N HCland extracted with ethyl acetate (2 × 100 ml.). The combined extractswere washed with water (30 ml.) and a saturated aqueous NaCl solution (2× 30 ml.), treated with a small amount of active carbon and dried overanhydrous Na₂ SO₄. The filtrate was evaporated to dryness to give 1.3 g.(92%) of 5 (R = H) as an oil.

ir: ν _(max) ^(liq) 3000-3600, 1670, 1260, 1150 cm⁻¹.

nmr: δ _(ppm) ^(CDCl).sbsp.3 1.44 (9H, s, C(CH₃)₃), 2.73 (3H, s,N--CH₃), 3.54 (2H, s, CH₂ CO), 4.38 (2H, s, CH₂ N), 6.5-7.3 (3H, m,phenyl-H).

Preparation of Ortho-N-methylaminomethyl-phenylacetic Acid. ##STR22##o-(p-Toluenesulfonylaminomethyl)phenylacetic Acid (2)

To a stirred solution of o-aminomethylphenylacetic acid hydrochloride(7.50 g., 37 m.mol.) and sodium hydroxide (4.74 g., 118 m.mol.) in water(100 ml.) was added p-toluenesulfonyl chloride (7.64 g., 40 m.mol.) inportions at 60° C. The mixture was stirred for 1 hour at the sametemperature and acidified with hydrochloric acid. The mixture wasextracted with ethyl acetate (4 × 50 ml.). The combined extracts werewashed with water, treated with a small amount of carbon and dried. Thesolvent was evaporated under reduced pressure and the residuecrystallized from ethyl acetate to afford 2 as colorless prisms. Yield,9.84 g. (84%). M.p. 155°-156° C.

ir: ν _(max) ^(nuj) 3300, 1705, 1335, 1170 cm⁻¹.

nmr: δ _(ppm) ^(DMSO-d).sbsp.6 2.38 (3H, s, CH₃), 3.65 (2H, s, CH₂ CO),3.97 (2H, d, J=5 Hz, CH₂ N), 7.1-8.2 (9H, m, phenyl-H & NH).

Anal. Calc'd. for C₁₆ H₁₇ NO₄ S: C, 60.17; H, 5.37; N, 4.39; S, 10.10.Found: C, 60.11, 60.15; H, 5.43, 5.40; N, 4.28, 4.30; S, 9.72, 9.80.

o-(N-p-Toluenesulfonyl-N-methylaminomethyl)phenylacetic Acid (3)

A mixture of 2 (9.0 g., 28 m.mol.) sodium hydroxide (6.0 g.) and methyliodide (6 ml.) in water (60 ml.) was heated in a sealed tube for 30minutes at 70° C. After cooling, the reaction mixture was acidified withhydrochloric acid to separate pale yellow precipitate which wascrystallized from ethyl acetate-n-hexane to give colorless prisms, 3.Yield, 8.5 g. (91%). M.p. 162°-163° C.

ir: ν _(max) ^(KBr) 2700-2300, 1700, 1600, 1345, 1200, 925 cm⁻¹.

nmr: δ _(ppm) ^(D).sbsp.2^(O+KOH) 2.37 (3H, s, CH₃), 2.49 (3H, s, CH₃),3.80 (2H, s, CH₂ CO), 4.18 (2H, s, CH₂ N), 7.0-8.0 (8H, m, phenyl-H).

Anal. Calc'd. for C₁₇ H₁₉ NO₂ : C, 61.24; H, 5.74; N, 4.20; S, 9.61.Found: C, 61.31, 61.36; H, 5.73, 5.71; N, 4.51, 4.29; S, 9.63, 9.55.

N-Methylaminomethylphenylacetic Acid (4)

Method A (using hydrobromic acid) -- A mixture of 28.6 g. (0.086 mol.)of 3 and 20 g. (0.213 mol.) of phenol in 260 ml. of 48% hydrobromic acidwas refluxed for 30 minutes. The mixture was cooled, diluted with thesame volume of water and washed with ethyl acetate (2 × 50 ml.). Theaqueous layer was evaporated to dryness in diminished pressure to givean oil which was chromatographed on a column of Amberlite IR-120 (H⁺form, 200 ml.) eluting with 5% ammonium hydroxide solution. The eluate(2.5 l.) was collected and evaporated to dryness under reduced pressure.The residue was triturated with acetone and crystallized from ethanol toafford 6.7 g. (43.5%) of 4 as colorless needles, melting at 168°-170° C.(dec.).

Method B (using metallic sodium in liquid ammonia) -- To a mixture of 3(35 g., 0.105 mol.) in liquid ammonia (1000 ml.) was added 13.3 g.(0.578 atom) of sodium in small pieces under vigorous stirring over aperiod of 2 hours. The ammonia was evaporated with stirring on awater-bath in a well-ventilated hood and finally under reduced pressureto remove it completely. The residue was dissolved in ice water (400ml.) and the solution was stirred with ion-exchange resin IRC-50 (H⁺form, 400 ml.) for 0.5 hour at room temperature. The resin was filteredoff and to the filtrate was added an aqueous 1 M solution of bariumacetate until no more precipitate was formed (ca 50 ml. of the bariumacetate solution was required). The mixture was filtered and thefiltrate was chromatographed on a column of IR-120 (H⁺ form, 400 ml.) asin Method A to give 13.6 g. (72%) of 4.

o-(N-methyl-N-t-butoxycarbonylaminomethyl)phenylacetic Acod (5)

t-Butyl 4,6-dimethylpyrimidin-2-ylthiolcarbonate (11 g., 0.048 mol.) wasadded in one portion to a mixture of 4 (7.2 g., 0.04 mol.) and1,1,3,3;-tetramethylguanidine (6.9 g., 0.06 mol.) in 50% aqueous THF andthe mixture was stirred overnight at room temperature. The THF beingevaporated under reduced pressure, the aqueous solution was acidified topH 2 with dil. hydrochloric acid and extracted with ethyl acetate (2 ×20 ml.). The combined extracts were washed with water, treated with asmall amount of active carbon and evaporated under diminished pressure.The residue was triturated with hexane and crystallized fromn-hexane-ether to afford 9.2 g. (83%) of 5 as colorless prisms. M.p.96°-98° C.

ir: ν _(max) ^(KBr) 1730, 1630, 1430, 1830, 1250 cm⁻¹.

nmr: δ _(ppm) ^(CDCl).sbsp.3 1.49 (9H, s, t-butyl), 2.78 (3H, s,N--CH₃), 3.72 (2H, s, CH₂ CO), 4.25 (2H, s, CH₂ N), 7.28 (4H, s,phenyl), 9.83 (1H, s, --COOH).

Anal. Calc'd. for C₁₅ H₂₁ NO₄ : C. 64.50; H, 7.58; N, 5.01. Found: C,64.69; H, 7.66; N, 4.89.

Preparation of 3-N-methylaminomethyl-2-thienylacetic Acid ##STR23##3-Aminomethyl-2-thienylacetic Acid δ-lactam (2)

Glacial acetic acid (140 ml.) was added dropwise with stirring to amixture of 2-ethoxycarbonylmethylthiophene-3-carboxaldehyde oxime (1)(41 g., 0.19 mole) and zinc dust (65.4 g., 1 mole) in methanol, and themixture was stirred under reflux for 4 hours. The mixture was cooled andinsolubles were removed by filtration and washed with methanol (3 × 50ml.). The filtrate was combined with the washings and evaporated invacuo to dryness, the residue being extracted with methanol (5 × 100ml.). The methanol extracts were combined and evaporated under reducedpressure. To the residue was added water (50 ml.) and the mixture wasadjusted to pH 10 with Na₂ CO₃ and extracted with chloroform (33 × 100ml.). The combined chloroform extracts were washed with water (10 ml.),dried over MgSO₄, and evaporated under reduced pressure. The residualoil (30 g.) was triturated with hot benzene (150 ml.). The colorlessneedles were collected by filtration and recrystallized from ethylacetate to give the lactam 2 (7.7 g., 26%), melting at 195°-196° C. UV:λ_(max) ^(MeOH) 232 nm (ε, 6500)

Anal. Calc'd. for C₇ H₇ NOS: C, 54.88; H, 4.61; N, 9.14; S, 20.93.Found: C, 55.04; H, 4.45; N, 9.13; S, 20.50.

3-N-Methylaminomethyl-2-thienylacetic Acid δ-lactam (3)

To a suspension of sodium hydride (50% in paraffin, 1.82 g., 38 m.moles)in absolute benzene (500 ml.) was added the lactam 2 (4.85 g., 32m.moles) with stirring under nitrogen atmosphere and the mixture wasrefluxed for 2 hours. Methyl iodide (22.7 g., 160 m.moles) was added inone portion at room temperature and the mixture was again refluxed for 2hours. Ice-water (50 g.) was added to the mixture and organic layer wasseparated. The aqueous layer was extracted successively with benzene (2× 50 ml.) and chloroform (50 ml.). The extracts were combined and driedon MgSO₄. The solvent was evaporated under reduced pressure. To theresidue was added a hot mixture of benzene-n-hexane (1:1, 100 ml.) torecover 2 as needles (2.02 g., 42%). The filtrate was evaporated and theresidue was crystallized from benzene-n-hexane to afford colorlessplates 3. Yield: 2.7 g. (51%). M.p. 98°-100° C.

ir: ν_(max) ^(nujol) 1620 cm⁻¹.

nmr: δ_(max) ^(CHCl).sbsp.3 3.15 (3H, s, N--CH₃), 3.72 (2H, t, J=3Hz,CH₂ CO), 4.53 (2H, t, J=3Hz, --CH₂ --N), 6.87 (1H, d, J=4.5Hz,thiophene-Hβ), 7.30 (1H, d, J=4.5 Hz, thiophene-Hα).

uv: λ_(max) ^(MeOH) 232 nm (ε, 6700)

Anal. Calc'd. for C₈ H₉ NOS: C, 57.46; H, 5.42; N, 8.38; S, 19.17.Found: C, 57.56; H, 5.26; N, 8.31; S, 19.3.

3-(N-Methylaminomethyl)-2-thienylacetic Acid (4)

A mixture of the lactam 3 (3.5 g., 21 m.moles) and 6N HCl (100 ml.) washeated under reflux for 12 hours. The mixture was treated with carbonand concentrated to dryness under reduced pressure. The residual oil wasdissolved in water (10 ml.) and chromatographed on a column of IR-120(H⁺, 50 ml.). The column was eluted with water (200 ml.) and 5N NH₄ OH(3 L.). The amino acid 4 (3.0 g., 77%) was isolated by evaporation ofthe ammonia eluates followed by crystallization from aqueous acetone.M.p. 181°-182° C.

ir: ν_(max) ^(KBr) 1570, 1360 cm⁻¹.

nmr: δ_(ppm) ^(D).sbsp.2^(O) 2.21 (3H, s, N--CH₃), 3.80 (2H, s, CH₂ CO),4.20 (2H, s, CH₂ --N), 7.19 (1H, d, J=6Hz, thiophene-Hβ), 7.46 (1H, d,J=6Hz, thiophene-Hα).

uv: λ_(max) ^(H).sbsp.2^(O) 237 nm (ε, 7600)

Anal. Calc'd. for C₈ H₁₁ NO₂ S: C, 51.87; H, 5.99; N, 7.56; S, 17.31.Found: C, 51.67; H, 6.50; N, 7.28; S, 16.69.

3-(N-t-butoxycarbonyl-N-methylaminomethyl)-2-thienylacetic Acid (5)

To a mixture of 3-N-methylaminomethyl-2-thienylacetic acid 4 (2.7 g.,14.6 m.moles) and triethylamine (6 g., 60 m.moles) in 50% aqueousacetone (60 ml.) was added dropwise t-butoxycarbonyl azide (4.2 g., 29.2m.moles) over a period of 20 minutes at 0° C. with vigorous stirring.The reaction mixture was stirred overnight at room temperature andconcentrated under reduced pressure. The concentrate was washed withether (2 × 20 ml.), adjusted to pH 2 with concentrated HCl and extractedwith ethyl acetate (2 × 50 ml.). The ethyl acetate extracts were washedwith a saturated aqueous NaCl solution, dried on MgSO₄, treated withcharcoal and evaporated under reduced pressure. The residue wastriturated with n-hexane and crystallized from n-hexane-benzene to give3.68 g. (88%) of colorless needles 5 melting at 82°-83° C.

ir: ν_(max) ^(nujol) 1730, 1640 cm⁻¹.

nmr: δ_(ppm) ^(CDCl).sbsp.3 1.47 (9H, s, BOC-H), 2.78 (3H, s, N--CH₃),3.87 (2H, s, CH₂ --CO), 4.48 (2H, s, CH₂ -N), 6.91 (1H, d, J-32 6Hz,thiophene-Hβ), 7.20 (1H, d, J=6Hz, thiophene-Hα), 10.63 (1H, s, CO₂ H,dissapeared by addition of D₂ O).

Anal. Calc'd. for C₁₃ H₁₉ NO₄ S: C, 54.72; H, 6.71; N, 4.91; S, 11.24.Found: C, 54.91; H, 6.85; N, 4.92; S, 11.19.

The use of an "en-amine" blocking group with a prospective 7-side chaincontaining a free amino group prior to acylation of a nucleus such as IIherein is well known as from U.S. Pat. Nos. 3,223,141, 3,813,390,3,813,391, 3,823,141 and Belgium Pat. No. 773,773.

Sodium 2-[N-(1-carbethoxypropen-2-yl)aminomethyl]-1.4-cyclohexadienylacetate (4)

To a stirred solution of 460 mg. (0.02 g. atom) of metallic sodium in100 ml. of absolute EtOH was added 3.34 g. (0.02 mole) of2-aminomethyl-1,4-cyclohexadienylacetic acid and 3.1 g. (0.024 mole ofethyl acetoacetate and the mixture was heated to reflux for 4 hours withstirring. The hot reaction mixture was filtered and the filtrate wasallowed to keep cold overnight to give 2.0 g. of colorless needles 4melting at 264° C. The additional product (3.3 g.) was obtained byconcentration of the mother liquid. The total yield was 5.3 g. (88%).

IR:ν_(max) ^(nuj) 3300, 1635, 1600, 1570, 1300, 1275, 1170, 1090 cm⁻¹.

NMR: δ_(ppm) ^(D).sbsp.2^(O) 1.23 (3H, t, 7Hz, CH₂ CH₃), 1.96 & 2.25(3H, s, C═C--CH₃, cis & trans), ##STR24## 3.04 (2H, s, CH₂ CO), 3.66 &3.95 (2H, s, CH₂ --N, cis & trans), 4.07 (2H, q, 7Hz, CH₂ CH₃),##STR25##

Anal. Calcd. for C₁₅ H₂₀ NO₄ Na: C, 59.79; H, 6.69; N, 4.64. Found: C,59.69; H, 6.76; N, 4.75.

2-t-Butoxycarbonylaminomethyl-4-hydroxyphenylacetic acid is prepared,for example, according to U.S. Pat. No. 3,823,141.

o-(N-Methylaminomethyl)phenylacetic acid δ-lactam ##STR26##

Sodium hydride (57% in paraffin, 4.3 g.; 0.11 mol.) was washed with dryn-hexane and suspended in dry benzene (100 ml.). To the suspension wasadded a solution of o-aminomethylphenylacetic acid δ-lactam (U.S.3,796,716) (14.7 g., 0.1 mol.) in dry benzene or xylene (200 ml.) withstirring under a nitrogen atmosphere. The mixture was refluxed for onehour and cooled to room temperature. To the mixture was added methyliodide (18 ml.) in one portion and the mixture was refluxed again for1.5 hours. The reaction mixture was cooled to room temperature andpoured into ice-water (100 ml.). The aqueous layer was separated fromthe organic layer and extracted with CHCl₃ (2 × 50 ml.). The extractswere combined with the organic layer and dried on MgSO₄. The solvent wasremoved and the oily residue was distilled in vacuo to afford 14.9 g.(92%) of o-(N-methylaminomethyl)phenylacetic acid δ-lactam, boiling at130°-135° C/2 mmHg., m.p. 35° -37° C.

ir: ν_(max) ^(KBr) 3300, 1620, 1490 cm⁻¹.

nmr: δ_(ppm) ^(CDCl).sbsp.3 3.12 (3H, s), 3.59 (2H, t, J=1.5 Hz), 4.48(2H, t, J=1.5 Hz), 7.21 (4H, br-s).

Anal. Calc'd. for C₁₀ H₁₁ NO.1/4H₂ O: C, 72.49; H, 6.84; N, 8.45. Found:C, 72.78, 72.70; H, 6.76, 6.81; N, 8.49, 8.51.

o-N-Methylaminomethylphenylacetic acid ##STR27##

A mixture of the above-produced o-(N-methylaminomethyl)phenylacetic acidδ-lactam (5.0 g., 0.031 mol) and conc hydrochloric acid (500 ml.) wasrefluxed for 40 hours. The mixture was evaporated under reducedpressure, and the residual oil was dissolved in water (20 ml.) andtreated with a small amount of active carbon. The filtrate was washedwith benzene (50 ml.) and evaporated to dryness. The residual oil wascrystallized by trituration with THF (or acetone) to give colorlesssneedles of o-N-methylaminomethylphenylacetic acid hydrochloride (4.5 g.,67%).

Anal. Calc'd. for C₁₀ H₁₃ NO₂.HCl: C, 55.69; H, 6.54; N, 6.49; Cl, 1644.Found: C, 55.65, 55.74; H, 6.62, 6.60; N, 6.53, 6.53; Cl, 16.36.

Some unreacted starting material was recovered from the benzene layerand the THF washings (1.2 g., 24%, b.p. 140°-143° C/2mmHg).

An aqueous solution of o-N-methylaminomethylphenylacetic acidhydrochloride (5 g.) was column chromatographed with IR-120 ion-exchangeresin (H⁺, 70 ml.) and eluted with 3N NH₄ OH (2 l) to afford 3.9 g.(93%) of o-N-methylaminomethylphenylacetic acid as needles.

ir: ν_(max) ^(KBr) 1650, 1470 cm⁻¹.

The following examples are given in illustration of, but not inlimitation of, the present invention. All temperatures are in degreesCentigrade. 7-Aminocephalosporanic acid is abbreviated as 7-ACA;-ACA-represents the moiety having the structure ##STR28## and thus 7-ACAcan be represented as ##STR29## Methyl isobutyl ketone is represented asMIBK. "Skellysolve B" is a petroleum ether fraction of B.P. 60°-68° C.consisting essentially of n-hexane.

LA-1 resin is a mixture of secondary amines wherein each secondary aminehas the formula ##STR30## wherein each of R¹, R² and R³ is a monovalentaliphatic hydrocarbon radical and wherein R¹, R² and R³ contain in theaggregate from eleven to fourteen carbon atoms. This particular mixtureof secondary amines, which is sometimes referred to in these examples as"Liquid Amine Mixture No. II," is a clear amber liquid having thefollowing physical characteristics: viscosity at 25° C. of 70 cpd.,specific gravity at 20° C. of 0.826; refractive index at 25° C. of1.4554; distillation range at 10 mm., up to 170° C. -- 0.5%, 170°-220°C. -- 3%, 220°-230° C. -- 90% and above 230° C. -- 6.5%.

IR-120 is also called Amberlite IR-120 and is a strong cation exchangeresin containing sulfonic acid radicals. Amberlite IR-120 is acommercially available cation exchange resin of the polystyrene sulfonicacid type; it is thus a nuclear sulfonated polystyrene resin cross-linedwith divinyl benzene obtained by the procedure given by Kunin, IonExchange Resins, 2nd. Edition (1958), John Wiley and Sons, Inc. Thereinsee pages 84 and 87 for example.

Amberlite IRC-50 is a commercially available cation exchange resin ofthe carboxylic type; it is a copolymer of methacrylic acid anddivinylbenzene.

Dicyclohexylcarbodiimide is abbreviated as DCC, tetrahydrofuran as THF,thin layer chromatography as TLC, p-toluenesulfonyl as Ts and methanolas MeOH.

When the following instrumental readings are given, for infrared nu ifused is written ν, for ultraviolet lambda is written as λ, with molarabsorptivity as epsilon (ε) and for nuclear magnetic resonance (nmr)delta is written as δand tau as τ (δ = 10-τ).

DESCRIPTION OF THE PREFERRED EMBODIMENTS Synthesis (Schemes 1, 2 and 3)

The 3-side chain thiol,2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiol(3), was prepared by N-ethoxycarbonylmethylation of6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-one (1) with sodiumhydride and ethyl chloroacetate in DMF (dimethylformamide) andsubsequent thiolation with sodium hydrosulfide (Scheme 1). Condensationof 7-ACA (7-aminocephalosporanic acid) with 3 carried out by refluxingin phosphate buffer (pH 7) to give the 3-substituted-thio 7-ACA (4),which was coupled with an appropriate N-BOC-protected amino acid by theactive ester method using 2,4-dinitrophenol (DNP). The resultingN-BOC-protected cephalosporins 7 and 11 were deblocked with TFA(trifluoroacetic acid) and converted to the monosodium salt with Nsodium hydroxide (Schemes 2 and 3).

    ______________________________________                                        Scheme 1. Preparation of 7-Amino-3-(2-carboxymethyl-                          2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-                         methyl)-3-cephem-4-carboxylic Acid.                                           ______________________________________                                         ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                     ##STR34##                                                                    ______________________________________                                    

    __________________________________________________________________________    Scheme 2.                                                                     Preparation of 7-[(o-Aminomethylphenyl)-acetamido]                            3-(2-carboxymethyl-2,3-dihydro-s-triazolo-[4,3-b]pyridazin-                   3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acids.                             __________________________________________________________________________     ##STR35##                                  5                                  ##STR36##                                  6                                  ##STR37##                                  7                                  ##STR38##                                  8                                          a: R.sup.1 = H, R.sup.2 =H                                                                    8a: BB-S469                                                   b: R.sup.1 = CH.sub.3, R.sup.2 = H                                                            8b: BB-S479                                                   c: R.sup.1 = CH.sub.3, R.sup.2 = OH                                                           8c: BB-S478                                          __________________________________________________________________________

    __________________________________________________________________________    Scheme 3.                                                                     Preparation of 7-[(3-Aminomethyl-2-thienyl-acetamido)                         3-(2-carboxymethyl-2,3-dihydro-s-triazolo-[4,3-b]pyridazin-                   3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acids.                             __________________________________________________________________________     ##STR39##                                9                                    ##STR40##                                10                                   ##STR41##                                11                                   ##STR42##                                12                                               a: R = H  12a: BB-S483                                                        b: R = CH.sub.3                                                                         12b: BB-S472                                           __________________________________________________________________________

6-Chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo[4,3-b]pyridazin-3-one(2)

To a solution of 6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-one[P. Francavilla and F. Lauria, J. Het. Chem., 8, 415 (1971)] (1, 1.00g., 5.9 m.mole) in dry DMF (30 ml.) was added sodium hydride (50% inparaffin, 0.3 g., 6.3 m.mole) under stirring with formation of yellowcrystals. To the mixture was added ethyl chloroacetate (1.4 ml., 13m.mole) and the mixture was heated at 90° C. for 8 hours with stirring.After cooling, the reaction mixture was poured into water (50 ml.) andextracted with toluene (5 × 40 ml.). The organic extracts were combined,dried over anhydrous sodium sulfate and evaporated at reduced pressure.The residue was crystallized with benzene-n-hexane to give yellowneedles of 2 (1.16 g., 77%), m.p. 114°-115° C. (lit. 110° C.).

ir: ν_(max) ^(KBr) 1735, 1710 cm⁻¹.

uv: λ_(max) ^(EtOH) 231 nm (ε, 26,000)

nmr: δ_(ppm) ^(CDCl).sbsp.3 7.58 (1H, d, J=10 Hz, pyridazine-H), 6.98(1H, d, J=10 Hz, pyridazine-H), 4.80 (2H, s, --CH₂ CO), 4.27 (2H, q,J=7.5 Hz, CH₂ CH₃), 1.29 (3H, t, J=7.5 Hz, CH₂ CH₃).

Anal. Calc'd for C₉ H₉ N₄ O₃ Cl: C, 42.12; H, 3.53; N, 21.83; Cl, 13.81.Found: C, 41.54, 41.46; H, 3.22, 3.49 N, 21.51, 21.53; Cl, 13.88, 13.99.

2-Carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo[4,3-b]pyridazin-3-one(3)

To a solution of6-chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo[4,3-b]pyridazin-3-one(2, 30 g., 0.12 mole) in ethanol (900 ml.) was added NaSH.2H₂ O (70%pure, 45.9 g., 0.36 mole) and the mixture was refluxed for 0.5 hour. Thereaction mixture was evaporated at reduced pressure. The residue wasdissolved in water (200 ml.) and concentrated HCl was added to thesolution to adjust to pH 2. The precipitate (3) was collected byfiltration and washed with water. Yield 18.3 g. (69%).

ir: ν_(max) ^(KB4) 2900, 2450, 1750, 1660 cm⁻¹.

uv: λ_(max) ^(1%NaHCO).sbsp.3^(aq). 260 nm (ε, 19,500), 313 nm (ε, 7000)

nmr: δ_(ppm) ^(DMSO-d).sbsp.6 7.88 (1H, d, J=10 Hz, pyridazine-H), 7.45(1H, d, J=10 Hz, pyridazine-H), 4.72 (2H, s, CH₂ CO).

Anal. Calc'd. for C₇ H₆ N₄ O₃ S: C, 37.17; H, 2.67; N, 24.77; S, 14.17.Found: C, 37.35, 37.23; H, 2.26, 2.28; N, 23.58, 23.69; S, 14.32.

7-Amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (4)

To a suspension of 7-aminocephalosporanic acid (8.79 g., 32.2 m.mole) in0.1 M phosphate buffer (pH 7, 149 ml.) were added NaHCO₃ (8.14 g., 97.0m.mole) and the thiol 3 (7.30 g., 32.2 m.mole) with stirrinng. Themixture was heated at 80° C. for 0.5 hour under N₂ stream. The mixturewas treated with active carbon and adjusted to pH 3 with concentratedHCl. The resulting precipitate was collected by filtration and washedwith water to give 7.59 g. (54%) of 4.

ir: ν_(max) ^(KBr) 1800, 1720, 1600, 1540, 1470 cm⁻¹.

uv: λ_(max) ^(Buffer) (pH 7) 252 nm (ε, 19,500), 298 nm (ε, 8400).

nmr: δ_(ppm) ^(D).sbsp.2^(O+K).sbsp.2^(CO).sbsp.3 7.56 (1H, d, J=9 Hz,pyridazine-H), 7.05 (1H, d, J=9 Hz, pyridazine-H), 5.45 (1H, d, J=5 Hz,6-H), 5.05 (1H, d, 5 Hz, 7-H), 4.43 (1H, d, J=14 Hz, 3-CH₂), 4.04 (1H,d, J=14 Hz, 3-CH₂), 3.88 (1H, d, J=18 Hz, 2-H), 3.45 (1H, d, J=18 Hz,2-H).

EXAMPLE 1 Preparation of BB-S469 ##STR43## 2,4-Dinitrophenylo-(N-butoxycarbonylaminomethyl)phenylacetate (6a)

To a mixture of o-(N-butoxycarbonylaminomethyl)phenylacetic acid (5a,13g., 49 m.mole) and 2,4-dinitrophenol (9.02 g., 49 m.mole) in dry ethylacetate (123 ml.) was added dicyclohexylcarbodiimide (DCC) (10.1 g., 49m. mole) under water cooling (5°-15° C.), and the mixture was stirredfor 30 minutes at room temperature. The resulting precipitate wasfiltered off and the filtrate was evaporated to give 23.9 g. of theactive ester 6a, which was used in the next acylation reaction withoutfurther purification.

ir: ν_(max) ^(KBr) 1775, 1700, 1600, 1530 cm⁻¹.

7-[o-(N-Butoxycarbonylaminomethyl)phenylacetamido]-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (7a)

To a cold (0° C.) mixture of 4 (4.38 g., 10 m.mole), Et₃ N (4.5 ml., 30m.mole), CH₃ CN (20 ml.) and water (20 ml.) was added a solution of2,4-dinitrophenyl o-(N-butoxycarbonylaminomethyl)phenylacetate (6a, 4.79g.) in THF (tetrahydrofuran) (20 ml.). After stirring at roomtemperature overnight, THF and CH₃ CN in the reaction mixture wereremoved at reduced pressure and the resulting aqueous solution wasadjusted to pH 2 with dilute HCl and extracted with ethyl acetate (10 ×30 ml.). The organic extracts were dried over sodium sulfate andevaporated. The residue was chromatographed on a column of silica gel(60 g.) and eluted with CHCl₃ and 3% MeOH-CHCl₃ successively to give2.40 g. (37%) of 7a, mp. >161° C. (dec.).

ir: ν_(max) ^(KBr) 1780, 1720 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 252 nm (ε, 19800), 298 nm (ε, 8900).

nmr: δ_(ppm) ^(DMSO+D).sbsp.2^(O) 7.67 (1H, d, J=9.0 Hz, pyridazine-H),7.10 (4H, s, phenyl-H), 7.05 (1H, d, J=9.0 Hz, pyridazine-H), 5.66 (1H,d, J=4.5 Hz, 7-H), 5.07 (1H, d, J=4.5 Hz, 6-H), 4.71 (2H s, N--CH₂--CO), 4.4-4.0 (4H, m, 3-CH₂ & CH₂ --N), 3.8-3.5 (4H, m, 2-H & CH₂ CO),1.42 (9H, s, t-Butyl-H).

Anal. Calc'd. for C₂₈ H₃₁ N₇ O₇ S₂.2H₂ O: C, 49.62; H, 5.20; N, 14.46;S, 9.46. Found: C, 49.97, 49.95; H, 4.79, 4.62; N, 14.00, 13.84; S,9.37, 9.32.

BB-S469;7-[(o-(Aminomethylphenyl)acetamido]-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (8a)

Trifluoroacetic acid (3.4 ml.) was added to 7a (2.33 g.) at 0° C. andthe mixture stirred for 15 minutes at room temperature. To the mixturewas added dry ether (100 ml.), and the precipitate was collected byfiltration and washed with dry ether (2 × 50 ml.). The solid wasdissolved in a mixture of CH₃ CN (100 ml.) and water (14 ml.) and thesolution was adjusted to pH 4-5 with concentrated NH₄ OH to afford aprecipitate which was collected by filtration and washed with CH₃ CN (2× 50 ml.) to give 1.75g. (82%) of 8a as the ammonium salt. M.p. >160° C.(dec.).

ir: ν_(max) ^(KBr) 1765, 1710, 1640, 1590 cm⁻¹.

uv. λ_(max) ^(pH) 7 Buffer 252 nm (ε, 23,900), 298 nm (ε, 10,900).

Anal. Calc'd. for C₂₄ H₂₂ N₇ O₇ S₂.NH₄ ⁺.H₂ O: C, 46.51; H, 3.96; N,18.08; S, 10.35. Found: C, 46.52, 46.26; H, 4.15, 4.19; N, 17.60, 1748;S, 10.89, 10.41.

Preparation of BB-S469 Monosodium Salt

To a suspension of 8a (1.58 g.) in 50% acetonewater (30 ml.) was added a10% solution to adjust the pH to 7.7. An additional amount of acetonewas added and the precipitate was collected by filtration and washedwith acetone to give 1.38 . (87%) of monosodium salt of 8a, m.p. >170°C. (dec.).

ir: ν_(max) ^(KBr) 1765, 1710, 1640, 1600, 1540, 1485 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 252 nm (ε, 20,600), 298 nm (ε, 9200).

nmr: δ_(ppm) ^(D).sbsp.2^(O+K).sbsp.2^(CO).sbsp.3 7.52 (1H, d, J=9 Hz,pyridazine-H), 7.27 (4H, s, phenyl-H), 7.03 (1H, d, J=9 Hz,pyridazine-H), 5.62 (1H, d, J=4.5 Hz, 7-H), 5.07 (1H, d, J=4.5 Hz, 6-H).

Anal. Calc'd for C₂₄ H₂₂ N₇ O₇ S₂ Na.2H₂ O: C, 44.79; H, 4.07; N, 15.23;S, 9.96. Found: C, 44.44, 44.95; h, 3.68, 3.90; N, 16.50, 16.67; S,10.38, 10.45.

EXAMPLE 2 Preparation of BB-S472 ##STR44##7-(3-N-t-Butoxycarbonyl-N-methylaminomethyl-2-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid. (11b)

A mixture of the BOX-protected amino acid (10b, 513 mg., 1.8 m.mole),2,4-dinitrophenol (400 mg., 2.16 m.mole) and DCC (445 mg., 2.16 m.mole)in THF (5 ml.) was stirred at room temperature for 12 hours. Theprecipitated urea was removed and the filtrate was added to a mixture of7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicacid (4, 800 mg., 1.8 m.mole) and triethylamine (0.76 ml., 5.4 m.mole)in water (5 ml.) at 0° C. with stirring. Stirring was continued untilactive ester had disappeared on tlc (silica gel plate; Rf 0.95; solventsystem, CHCl₃ :MeOH=3:1). The reaction mixture was diluted with water(20 ml.), layered with AcOEt (50 ml.) and adjusted to pH 2 withconcentrated HCl at 5° C. The organic layer was separated and aqueouslayer was extracted with AcOEt (3 × 50 ml.). The AcOEt extracts werecombined, washed with sat. aq. NaCl, dried over MgSO₄ and concentratedunder reduced pressure. The residual oil (1.9 g.) was chromatographed onsilica gel (40 g.). The column was eluted successively with CHCl₃ (400ml.), 3% MeOH--CHCl₃ (100 ml.) and 10% MeOH-CHCl₃ while monitoring withtlc (silica gel plate, solvent system MeOH:CHCl₃ =1:2, detected withI₂). From the CHCl₃ eluate was recovered a mixture of 2,4-DNP and theBOC-protected amino acid 9 and from 3% MeOH-CHCl₃ eluate 50 mg. of 9.The desired product (11b) (Rf 0.4, solvent system CHCl₃ -MeOH=3:1) wasobtained by evaporation of the eluate with 10% MeOH-CHCl₃. Yield 490 mg.(39%). M.p. 215°-220° C.

ir: ν_(max) ^(KBr) 3400, 1780, 1720, 1680, 1550 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 245 nm (ε, 23,000), 260 nm (ε, 18,000), 300nm (ε, 7900).

nmr: δ_(ppm) ^(DMSO-d).sbsp.6 1.42 (9H, s, BOC-H), 2.75 (3H, s, N--CH₃),##STR45## 4.72 (2H, s, BOC--N--CH₂), 5.10 (1H, d, J=4.5 Hz, 6-H), 5.70(1H, d-d, J=4.5 & 10.5 Hz, changed to a doublet J=4.5 by addition of D₂O, 7-H), 6.85 (1H, d, J=4.5 Hz, thiophene Hβ), 7.19 (1H, d, J=9 Hz,pyridazine H), 7.34 (1H, d, J=4.5 thiophene Hα), 7.72 (1H, d, J=9 Hz,pyridazine-H), 9.11 (1H, d, J=10.5 Hz, disappeared by addition of D₂ O,NH).

Anal. Calc'd for C₂₈ H₃₁ N₇ O₉ S₃.H₂ O: C, 46.46; H, 4.60; N, 13.55; S,13.29. Found: C, 46.67; H, 4.71; N, 12.79; S, 12.81.

BB-S472;7-(3-Methylaminoemethyl-2-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (12b)

Trifluoroacetic acid (0.4 ml.) was added to 11b (400 mg., 0.57 m.mole)at 0° C. and the mixture was stirred at room temperature for 15 minutes.To the reaction mixture was added anhydrous ether (10 ml.) to separate aprecipitate which was collected by filtration, washed with anhydrousether (2 × 10 ml.) and suspended in acetonitrile (10 ml.). Thesuspension was adjusted to pH 4 with concentrated NH₄ OH and stirred for10 minutes. The solid was collected by filtration washed withacetonitrile (2 × 5 ml.) and dried at 60° C./1mmHg for 7 hours to afford310 mg. (90%) of 12b melting at 188°-191° C. (dec.).

ir: ν_(max) ^(KBr) 3400, 1770, 1720, 1680, 1550 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 245 nm (ε, 22,400), 260 nm (68, 18,700), 300nm (ε, 8600).

Anal. Calc'd. for C₂₃ H₂₃ N₇ O₇ S₃.3H₂ O: C, 41.87; H, 4.43; N, 14.86;S, 14.58. Found: C, 42.03; H, 3.59; N, 14.79; S, 14.35.

Preparation of BB-S472 Monosodium Salt

To a suspension of 12b (230 mg., 0.38 m.mole) in 0.5 ml. of deionizedwater was added N NaOH to adjust to pH 8.9. Acetone (15 ml.) was addedto the solution. The precipitate was collected by filtration, washedwith acetone (2 × 5 ml), and dried at 60° C./1mmHg for 7 hours to afford170 mg. (71%) of monosodium salt of BB-S472, m.p. >210° C. (dec.). ir:ν_(max) ^(KBr) 3400, 1765, 1710, 1680, 1600 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 245 nm (ε, 21800), 260 nm (ε, 18500), 300 nm(ε, 7800).

nmr: δ_(ppm) ^(D).sbsp.2^(O) 2.72 (3H, s, N-CH₃), 3.45 (1H, d, J=18 Hz,2-H), 3.75 (1H, d, J=18 Hz, 2-H), ##STR46## 4.57 (2H, s, N--CH₂), 5.00(1H, d, J=4.5 Hz, 6-H), 5.53 (1H, d, J=4.5 Hz, 7-H), 6.97 (1H, d, J=9Hz, pyridazine-H), 7.03 (1H, d, J=4.5 Hz, thiopene-Hβ), 7.34 (1H, d,J=4.5 Hz, thiophene-Hα), 7.48 (1H, d, J=9 Hz, pyridazine-H).

Anal. Calc'd. for C₂₃ H₂₂ N₇ O₇ S₃ Na.1/2H₂ O: C, 43.40; H, 3.64; N,15.40; S, 15.11. Found: C, 43.26; H, 4.08; N, 14.18; S, 13.91.

EXAMPLE 3 Preparation of BB-S478 ##STR47##7-[2-(N-t-Butoxycarbonyl-N-methylaminomethyl)-4-hydroxyphenylacetamido]-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,5-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (7c)

A mixture of2-N-t-butoxycarbonyl-N-methylaminomethyl-4-hydroxyphenylacetic acid(5c), (708 mg., 2.4 m.mole), 2,4-dinitrophenol (478 mg., 2.6 m.mole) andDCC (536 mg., 2.6 m.mole) in dry THF (20 ml.) was stirred at roomtemperature for 2 hours. The precipitated urea was removed byfiltration. The filtrate was added to a solution of7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,5-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicacid (4) (876 mg., 2 m.mole) in 20 ml. of water containing triethylamine(0.84 ml., 6 m.mole) and the mixture was stirred at room temperature for18 hours. After concentrating to 20 ml. the aqueous solution was washedwith ether, acidified with 6N HCl and extracted with 200 ml. of ethylacetate. The extract was filtered to remove insolubles, washed withwater and a saturated aqueous NaCl solution and dried. The solution wasevaporated to dryness and the oily residue was chromatographed on asilica gel (Wakogel C-200, 25 g.) eluting with chloroform and 3% ofchloroform-methanol. The fractions containing the desired product(monitored by tlc; Rf 0.3; solvent system, CHCl₃ :MeOH=2:1) werecollected and evaporated to dryness. The oily residue was trituratedwith ether-n-hexane to give 630 mg. (44%) of the product 7c melting at200°-210° C. (slow dec.).

ir: ν_(max) ^(KBr) 1780, 1720, 1660, 1400, 1240, 1150 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 252 nm (ε, 13,000), 300 nm (ε, 5400).

nmr: δ_(ppm) ^(DMSO-d).sbsp.6 1.39 (9H, s, C--CH₃), 2.73 (3H, s,N--CH₃), 3.3-3.9 (4H, m, CH₂ CO & 2-H), 4.35 (4H, m, CH₂ N & 3-H), 4.48(2H, s, NCH₂ CO), 5.03 (1H, d, 4.5 Hz, 6-H), 5.61 (1H, d-d, 8 & 4.5 Hz,7-H), 6.4-7.2 (3H, m, phenyl-H), 6.98 (1H, d, 10 Hz, pyridazine-H), 7.61(1H, d, 10 Hz, pyridazine-H), 8.87 (1H, d, 8 Hz, NH).

Anal. Calc'd. for C₃₀ H₃₃ N₇ O₁₀ S₂ : C, 50.34; H, 4.65; N, 13.70; S,8.96. Found: C, 50.98; H, 5.36; N, 11.88; S, 7.60.

BB-S478;7-(2-N-Methylaminomethyl-4-hydroxyphenylacetamido)-3-(2-N-carboxymethyl-2,3-dihydro-s-triazolo[4,5-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (8c)

A mixture of 7c (570 mg., 0.8 m.mole) and trifluoroacetic acid (1.5 ml.)was stirred at 10° C. for 30 minutes and the mixture was diluted withether (50 ml.) to separate the trifluoroacetate of 8c which wascollected by filtration and then dissolved in a mixture of 10 ml. ofacetonitrile and 5 ml. of water and then filtered. The filtrate wasadjusted to pH 6 with concentrated ammonium hydroxide and the mixturewas diluted with acetonitrile (100 ml.). The resulting precipitate wascollected by filtration, washed with acetonitrile and dried in vacuoover P₂ O₅ to give 370 mg. (75%) of 8c, melting at 215°-220° C. (dec.).

ir: ν_(max) ^(KBr) 1770, 1710, 1600, 1380, 1350 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 252 nm (ε, 19,000), 300 nm (ε, 9100).

nmr: δ_(ppm) ^(D).sbsp.2^(O+K).sbsp.2^(CO).sbsp.3 2.75 (3H, s, N--CH₃),2.9-3.3 (4H, m, CH₂ CO & 2-H), 4.0-4.3 (4H, m, CH₂ N & 3-H), 4.57 (2H,s, NCH₂ CO), 4.81 (1H, d, 4.5 Hz, 6-H), 5.53 (1H, d, 4.5 Hz, 7-H),6.6-7.5 (5H, m, phenyl-H & pyridazin-H).

Anal. Calc'd. for C₂₅ H₂₅ N₇ O₈ S₂.5/2H₂ O: C, 45.45; H, 4.58; N, 14.84;S, 9.71. Found: C, 45.69; H, 4.21; N, 15.03; S, 9.46.

Preparation of Monosodium Salt of BB-S478

To a suspension of 8c (308 mg., 0.5 m.mole) in water (2ml.) was added0.3-0.4 ml. of N NaOH and the mixture was stirred at room temperature;the pH of the resulting solution was 9.2. Acetone (20 ml.) was slowlyadded to the solution. The resulting precipitate was collected byfiltration, washed with acetone (10 ml.) and dried in vacuo over P₂ O₅to give 290 mg. (91%) of the monosodium salt of BB-S478, melting at230°-235° C. (dec.).

ir: ν_(max) ^(KBr) 1770, 1700, 1600, 1390, 1350 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 250 nm (ε, 18,000), 300 nm (ε, 8400).

Anal. Calc'd. for C₂₅ H₂₄ N₇ O₈ S₂ Na.5/2H₂ O: C, 43.98; H, 4.28; N,14.36; S, 9.39. Found: C, 43.96; H, 4.14; N, 13.51; S, 9.34.

EXAMPLE 4 Preparation of BB-S479 ##STR48##7-[o-(N-Butoxycarbonyl-N-methylaminomethyl)phenylacetamido]-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (7b)

To a cold (0° C.) mixture of 4 (5.4 g., 12 m.mole), Et₃ N (5.5 ml.), CH₃CN (25 ml.) and water (25 ml.) was added a solution of 2,4-dinitrophenylo-(N-butoxycarbonyl-N-methylaminomethyl)phenylacetate (6b) in THF[prepared from o-(N-butoxycarbonyl-N-methylaminomethyl)phenylacetic acid(5b) (3.48 g., 13.5 m.mole), 2,4-dinitrophenol (2.49 g., 13.5 m.mole)and DCC (2.79 g., 13.5 m.mole) in dry THF (37 ml.)]. The mixture wasstirred at room temperature overnight. THF and CH₃ CN were removed fromthe reaction mixture by evaporation under reduced pressure and theresulting aqueous solution was washed with ether (3 × 30 ml.), adjustedto pH 2-3 with dilute HCl and extracted with ethyl acetate (4 × 30 ml.).The organic extracts were combined, dried over sodium sulfate andevaporated. The residue was chromatographed on a column of SiO₂ (100g.). After washing with CHCl₃, the column was eluted with 3% MeOH inCHCl₃ to afford a desired fraction containing 7b. Yield 3.8 g. (45%).m.p. >200° C. (dec.).

ir: ν_(max) ^(KBr) 1780, 1720, 1680 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 250 nm (ε, 18,800), 297 nm (ε, 8400).

nmr: δ_(ppm) ^(DMSO+D).sbsp.2^(O) 7.62 (1H, d, J=10.5 Hz, pyridazine-H),7.14 (4H, s, phenyl-H), 6.98 (1H, d, J=10.5 Hz, pyridazine-H), 5.61 (1H,d, J=4.5 Hz, 7-H), 5.03 (1H, d, J=4.5 Hz, 6-H), 4.67 (2H, s, N--CH₂),4.42 (2H, s, CH₂ --N), 4.4-4.0 (2H, m, 3--CH₂), 3.8--3.4 (4H, m, 2-H &CH₂ --CO), 2.72 (3H, s, N--CH₃), 1.38 (9H, s, BOC--H).

Anal. Calc'd. for C₃₀ H₃₃ N₇ O₉ S₂.5/2H₂ O: C, 48.38; H, 5.14; N, 13.16;S, 8.61. Found: C, 48.25, 48.23; H, 4.52, 4.46; N, 12.93, 12.86; S,8.68.

BB-S479;7-[o-(Methylaminomethyl)phenylacetamido]-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (8b)

Trifluoroacetic acid (7 ml.) was added to the t-BOC-derivative 7b (3.8g., 5.5 m.mole) at 0° C., and the mixture was stirred for 20 minutes atroom temperature. Dry ether (100 ml.) was added to the mixture. Theresulting precipitate was collected by filtration and washed with dryether (3 × 100 ml.). The precipitate was dissolved in a mixture of CH₃CN (120 ml.) and water (18 ml.) and the solution was adjusted to pH 5-6with concentrated NH₄ OH to give an oily precipitate which wastriturated with CH₃ CN to form solid material. The product 8b wascollected by filtration, washed with CH₃ CN and dried. Yield 2.55 g.(77%).

ir: ν_(max) ^(KBr) 1770, 1710, 1600, 1550 cm⁻¹.

Preparation of Monosodium Salt of BB-S479

To a solution of BB-S479 (8b) (2.54 g., 4.3 m.mole) in water (25 ml.), NNaOH (ca. 3 ml.) was added under cooling (the pH of the solution was10). A large amount of acetone was added to the solution and theprecipitate was collected by filtration and washed with acetone to give1.94 g. (84%) of monosodium salt of BB-S479. M.p. >200° C. (dec.).

ir: ν_(max) ^(KBr) 1770, 1710, 1600, 1550 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 250 nm (ε, 19,400), 297 nm (ε, 8700).

Anal. Calc'd. for C₂₅ H₂₄ N₇ O₇ S₂ Na.1/2H₂ O: C, 47.61; H, 4.00; N,15.55; S, 10.17. Found: C, 47.43, 47.43; H, 4.67, 4.68; N, 15.97, 15.70;S, 9.25, 9.84.

EXAMPLE 5 Preparation of BB-S483 ##STR49##7-(3-N-t-Butoxycarbonylaminomethyl-2-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (11a)

A mixture of the BOC-protected amino acid (9, 410 mg., 1.56 m.mole),2,4-dinitrophenol (313 mg., 1.7 m.mole) and DCC (353 mg., 1.7 m.mole) inTHF (5ml.) was stirred at room temperature for 12 hours. Theprecipitated urea was removed and the filtrate was added to a mixture of7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicacid (4, 683 mg., 1.56 m.mole) and triethylamine (0.62 ml., 4.68 m.mole)in water (5 ml.) at 0° C. with stirring. Stirring was continued at roomtemperature until the active ester disappeared on tlc (silica gel plate;Rf 0.95; solvent system, CHCl₃ :MeOH=3:1). The reaction was diluted withwater (20 ml.), layered with AcOEt (50 ml.) and adjusted to pH 2 withconcentrated HCl at 5° C. The organic layer was separated and theaqueous layer was extracted with AcOEt (3 × 50 ml.). The AcOEt (ethylacetate) exracts were combined, washed with saturated aqueous NaCl,dried over MgSO₄ and concentrated under reduced pressure. The residualoil (1.8 g.) was chromatographed on silica gel (40 g.). The column waseluted successively with CHCl₃ (400 ml.) and 3% MeOH--CHCl₃ (500 ml.).The eluate was monitored with tlc (silica gel plate, solvent systemCHCl₃ :MeOH=2:1, detected with I₂). The desired product 11a (Rf 0.2) wasobtained by evaporation of the MeOH--CHCl₃ eluate. Yield 450 mg. (42%),melting at 155°-160° C.

ir: ν_(max) ^(KBr) 3300, 1775, 1720, 1680 cm⁻¹.

uf: λ_(max) ^(pH) 7 Buffer 245 nm (ε, 23,900), 260 nm (ε, 19,200), 300nm (ε, 8700).

nmr: δ_(ppm) ^(DMSO-d).sbsp.6 1.39 (9H, s, BOC--H), ##STR50## 4.05 (2H,d, J=6 Hz, changed to a singlet by addition of D₂ O, BOCNH--CH₂), 4.20(2H, m, 3--CH₂), 4.69(2H, s, N--CH₂ CO₂), 5.06 (1H, d, J=4.5 Hz, 6-H),5.62 (1H, d-d, J=4.5 & 9 Hz, changed to a doublet J=4.5 Hz by additionof D₂ O, 7-H), 6.83 (1H, d, J=4.5 Hz, thiophene-Hβ), 7.00 (1H, m,disappeared by addition of D₂ O, NHBOC), 7.04 (1H, d, J=9 Hz,pyradizine-H), 7.12 (1H, d, J=4.5 Hz, thiophene-Hα), 7.65 (1H, d, J=9Hz, pyridazine-H), 8.97 (1H, d, J=9 Hz, disappeared by addition of D₂ O,7-NH).

Anal. Calc'd. for C₂₇ H₂₉ N₇ O₉ S₃ : C, 46.88; H, 4.23; N, 14.17; S,13.90. Found: C, 46.42; H, 4.37; N, 13.49; S, 13.61.

BB-S 483;7-(3-Aminomethyl-2-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (12a)

Trifluoroacetic acid (0.4 ml.) was added to 11a (410 mg., 0.59 m.mole)at 0° C. and the mixture was stirred at room temperature for 15 minutes.To the reaction mixture was added anhydrous ether (10 ml.) to separate aprecipitate which was collected by filtration, washed with anhydrousether (2 × 10 ml.) and suspended in acetonitrile (10 ml.). Thesuspension was adjusted to pH 4 with concentrated NH₄ OH and stirred for10 minutes. The precipitate was collected by filtration, washed withacetonitrile (2 × 5 ml.) and dried at 60° C./1mmHg for 7 hours to afford310 mg. (88%) of 12a, melting at above 200° C. (slow dec.).

ir: ν_(max) ^(KBr) 3400, 3150, 1760, 1700, 1680, 1600 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 245 nm (ε, 17,100), 260 nm (ε, 14,100), 300nm (ε, 6500).

Anal. Calc'd. for C₂₂ H₂₁ N₇ O₇ S₃.3H₂ O: C, 40.90; H, 4.21; N, 15.17;S, 14.89. Found: C, 40.39; H, 3.62; N, 15.87; S, 14.35.

Preparation of Monosodium Salt of BB-S483

To a suspension of 12a (280 mg., 0.47 m.mole) in 0.5 ml. of deionizedwater was added N NaOH to adjust to pH 9.5 and insoluble material wascollected by filtration. Acetone (15 ml.) was added to the filtrate toseparate the precipitate which was collected by filtration, washed withacetone (2 × 5 ml.) and dried at 70° C./1mmHg for 7 hours to afford 220mg. (76%) of monosodium salt of 12a. M.p. >210° C. (slow dec.).

ir: ν_(max) ^(KBr) 3400, 3250, 1760, 1710 1650, 1600, 1550 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 245 nm (ε, 19,900), 260 nm (ε, 16,400), 300nm (ε, 6900).

nmr: δ_(ppm) ^(D).sbsp.2^(O) 3.60 (2H, m, 2-H), 3.91 (2H, s, CH₂ CO),4.12 (2H, s, CH₂ --NH₂), 4.20 (2H, m, 3--CH₂), 4.55 (2H, s, N--CH₂ CO),4.95 (1H, d, J=4.5 Hz, 6-H), 5.50 (1H, d, J=4.5 Hz 7-H), 6.94 (1H, d,J=9 Hz, pyridazine-H), 6.99 (1H, d, J=4.5 Hz, thiophene-Hβ), 7.28 (1H,d, J=4.5 Hz, thiophene-Hα), 7.32 (1H, d, J=9 Hz, pyridazine-H).

Anal. Calc'd. for C₂₂ H₂₀ N₇ O₇ S₃ Na.1/2CH₃ COCH₃ : C, 43.92; H, 3.61;N, 15.26; S, 14.97. Found: C, 43.48; H, 4.56; N, 15.28; S, 13.91.

Solubility

All of the monosodium salts in this series showed more than 10%solubility in water.

Nephrotoxicity

A preliminary nephrotoxicity study was carried out by administration ofthe test compound to a group of two rabbits at 100 mg./kg.intravenously. The results obtained with BB-S469 and BB-S479 indicatedthat they might have little nephrotoxic potential.

In vitro Activity (Table 1)

The MIC's were determined by the serial dilution method usingMueller-Hinton agar against 51 gram-positive and 96 gram-negativebacteria. The 147 test organisms were classified into 16 groupsaccording to the genera and the types of antibiotic resistance, 5 groupsfor gram-positive and 11 for gram-negative bacteria. In Table 1 is shownthe in vitro activity in terms of geometric mean of MIC's. BB-S472 andBB-S479 showed better overall activity than their non-N-methylatedanalogs, BB-S483 and BB-S469, respectively. BB-S479 was superior toBB-S472 in some species of gram-negative bacteria. Comparing withcefamandole, BB-S479 was more active against most of the test organismsexcept against Providencia species and Staphylococci.

                                      TABLE 1                                     __________________________________________________________________________    In vitro Activity Against 147 Test Organisms                                               Geometric Mean MIC* (mcg./ml.)                                   __________________________________________________________________________                 No. of                                                                            BB-S469                                                                            BB-S472                                                                            BB-S478                                                                            BB-S479                                                                            BB-S483                                                                            Cefam-                              Test Organism                                                                              Strains                                                                           (Ex. 1)                                                                            (Ex. 2)                                                                            (Ex. 3)                                                                            (Ex. 4)                                                                            (Ex. 5)                                                                            andole                                                                            BL-S786**                       __________________________________________________________________________    S. aureus (sensitive)                                                                      4   0.4  0.4  0.4  0.3  0.3  0.1 1.3                             S. aureus (penicil-                                                            linase +)   13  1.2  0.9  1.0  0.9  1.1  0.4 3.0                             S. aureus                                                                      (Methicillin-R)                                                                           15  30   58   50   35   48   2.4 48                              S. pyogenes  10  0.05 0.05 0.04 0.04 0.08 0.04                                                                              0.2                             D. pneumoniae                                                                              9   0.03 0.05 0.03 0.04 0.04 0.2 0.08                            E. coli (sensitive)                                                                        13  0.3  0.2  0.2  0.2  0.3  0.3 0.4                             E. coli                                                                        (Penicillinase +)                                                                         7   11   15   10   2.6  21   5.7 9.3                             Enterobacter                                                                   (sensitive) 3   1.0  1.3  0.8  0.5  1.3  1.0 1.3                             Enterobacter                                                                   (Cephalosporinase +)                                                                      7   4.7  3.2  2.6  2.1  5.2  4.7 7.7                             Proteus (indole -)                                                                         6   0.6  0.3  0.4  0.5  0.7  0.6 0.9                             Proteus (indole + )                                                                        14  0.5  0.3  0.3  0.2  0.4  0.5 0.7                             Proteus (indole +,                                                             (cephalosporinase +)                                                                      5   11   8.3  7.3  4.8  17   3.6 29                              Providencia sp.                                                                            5   4.2  4.8  4.8  4.2  2.1  0.7 1.4                             Klebsiella sp.                                                                             12  0.9  0.8  0.6  0.6  1.1  2.4 0.8                             S. marcescens                                                                              16  130  200  40   35   81   34  180                             Miscellaneous (Sal-                                                            monella, Shigella,                                                            Citrobacter)                                                                              8   0.7  0.4  0.3  0.4  0.9  0.3 0.7                             __________________________________________________________________________     *Mueller-Hinton Agar (inoculum size: 10.sup.4 dilution) MIC cut-off: 200      mcg./ml.                                                                      **BL-S786 is sodium                                                           7-(2-aminomethylphenylacetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethy    )-3-cephem-4-carboxylate                                                  

EXAMPLE 6

Substitution in the procedure of Example 3 for the2-N-t-butoxycarbonyl-N-methylaminomethyl-4-hydroxyphenylacetic acid usedtherein of an equimolar weight of2-N-t-butoxycarbonylaminomethyl-4-hydroxyphenylacetic acid and of2-N-t-butoxycarbonylaminomethyl-4-methoxyphenylacetic acid and of2-N-t-butoxycarbonyl-N-methylaminomethyl-4-methoxyphenyacetic acid,respectively, produces the compounds having the structures ##STR51##

EXAMPLE 7 Preparation of BB-S493 ##STR52##7-[(2-N-t-Butoxycarbonylaminomethyl-1,4-cyclohexadienyl)acetamido]-3-(2-N-carboxymethyl-s-triazolo[4,5-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (2)

A mixture of 2-N-t-butoxycarbonylaminomethyl-1,4-cyclohexadienylaceticacid (1, 640 mg., 2.4 m.mole), 2,4-dinitrophenol (422 mg., 2.4 m.mole)and DCC (494 mg., 2.4 m.mole) in 10 ml. of dry THF was stirred for 1.5hours at room temperature. The precipitated urea was removed byfiltration. The filtrate was added in one portion to a solution of7-amino-3-(2-N-carboxymethyl-s-triazol[4,5b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicacid in 10 ml. of water containing triethylamine (0.56 ml., 4m.mole) andthe mixture was stirred at room temperature for 3 hours. The reactionmixture was concentrated to 10 ml. under reduced pressure, washed withether (3 × 10 ml.), acidified with 6N hydrochloric acid and extractedwith ethyl acetate (5 × 10 ml.). The combined extracts were washed witha saturated saline solution and dried with anhydrous Na₂ SO₄. Thesolvent was evaporated and the residue was chromatographed on silicagel(Wako-gel C-200, 30 g.) eluting with chloroform-methanol (0-50%). Thefractions containing the desired product were collected. The solvent wasremoved and the residue was triturated with ether-n-hexane to give 410mg. (30%) of the product 2. M.p. 110° C (decomp.).

ir: ν _(max) ^(KBr) 1780, 1730, 1610, 1530, 1250, 1160 cm⁻¹.

uv: ν _(max) ^(Buff) (pH 7) 252 nm (ε, 19000), 300 nm (ε, 8600, sh).

Anal. Calc'd. for C₂₉ H₃₃ N₇ O₉ S₂.2H₂ O: C, 48.12; H, 5.15; N, 13.54;S, 8.86. Found: C, 48.07; H, 4.64; N, 12.70; S, 8.39.

BB-S493;7-[(2-Aminomethyl-1,4-cyclohexadienyl)acetamido[-3-(2-N-carboxymethyl-s-triazolo[4,5-]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylicAcid (3)

The N-BOC-protected cephalosporin 2 (350 mg., 0.51 m.mole) was treatedwith trifluoroacetic acid (TFA) (1 ml.) for 30 minutes at roomtemperature. To the mixture was added ether (50 ml.) to give the TFAsalt of 3, which was collected by filtration and then dissolved in amixture of acetonitrile (5 ml.) and water (2 ml.). The solution wastreated with a small amount of active carbon, adjusted to pH 6 withconcentrated ammonium hydroxide. The precipitate was collected, washedwith acetonitrile (5 ml.) and dried in vacuo to afford 235 mg. (78%) of3. M.p. 220°-230° C. (decomp.).

ir: ν _(max) ^(KBR) 1770, 1740, 1710, 1650, 1600, 1550 cm⁻¹.

uv: λ _(max) ^(Buff) (pH 7) 252 nm (ε, 20,000), 300 nm (ε, 9000, sh).

Anal. Calc'd. for C₂₄ H₂₅ N₇ O₇ S₂.H₂ O: C, 47.60; H, 4.49; N, 16.19; S,10.59. Found: C, 47.77; H, 4.06; N, 16.49; S, 10.64.

    ______________________________________                                        In vitro Antibacterial Activity of BB-S493 Compared with                      BB-S479 and Cefamandole (Determined by Steers' Agar Dilu-                     tion Method on Mueller-Hinton Agar Plate)                                     ______________________________________                                                      MTC (meg./ml.)                                                  ______________________________________                                        Organism     BB-S493     BB-S479  Cefamandole                                 ______________________________________                                        S. aureus Smith                                                                           A9537   0.4      0.4    0.1                                       S. aureus   A9497   0.2      0.1     0.05                                     S. aureus BX-1633                                                                         A9606   0.4      0.4    0.2                                       St. faecalis                                                                              A9536   100      100     50                                       E. coli NIHJ                                                                              0.1      0.05     0.025                                           E. coli ATCC 8739   0.2       0.05   0.05                                     E. coli Juhl                                                                              A15119  0.2      0.1    0.4                                       E. coli BX-1373     0.4      0.2    0.4                                       E. coli     A15810  0.2      0.1    0.2                                       E. coli     A9660   0.1       0.05  0.1                                       E. coli     A15147  6.3      3.1    3.1                                       Kl. pneumoniae                                                                            A9678   0.4      0.4    1.6                                       Kl. pneumoniae                                                                            A9977   0.2      0.1    0.4                                       Kl. pneumoniae                                                                            A15130  0.2      0.1    0.4                                       Kl. pneumoniae                                                                            A9867   0.2      0.1    0.8                                       Pr. vulgaris                                                                              A9436   0.4      0.1    0.2                                       Pr. vulgaris                                                                              A9699   6.3      0.8     25                                       Pr. mirabilis                                                                             A9554   0.2      0.1    0.8                                       Pr. mirabilis                                                                             A9900   0.2      0.1    0.8                                       Pr. morganii                                                                              A9553   >100     >100   >100                                      Pr. morganii                                                                              A20031  0.4      0.1    0.8                                       Pr. rettgeri                                                                              A15167  0.1      0.1    0.1                                       Ps. aeruginosa                                                                            A9930   >100     >100   >100                                      Ps. aeruginosa                                                                            A9843   >100     >100   >100                                      Shig. dysenteriae    0.05     0.025 0.2                                       Shig. flexneri                                                                            A9684    25      12.5   3.1                                       Shig. sonnei                                                                              A9516    0.05     0.025  0.05                                     Serr. marcescens                                                                          A20019  100       25     50                                       Enterob. cloacae                                                                          A9656   6.3      1.6    3.1                                       Sal. enteritidis                                                                          A9531   0.1      0.05   0.1                                       Sal. typhosa                                                                              A9498   0.1      0.05   0.1                                       B. anthracis                                                                              A9504     0.0125  0.025 0.2                                       ______________________________________                                    

    __________________________________________________________________________    In vivo Activity of BB-S479 and Related Compounds (Mice, sc)                          (PD.sub.50 (mgm./kg.)                                                 __________________________________________________________________________    Organism                                                                              BB-S469                                                                            BB-S472                                                                            BB-S478                                                                            BB-S479                                                                            BB-S483                                                                            BB-S493                                                                            Cefamandole                                                                          BL-S786                          __________________________________________________________________________    S. aureus Smith                                                                       0.19 0.12 0.08 0.16 0.34 0.29 0.93   0.53                                     0.16           0.12           0.74   0.55                                     0.29           0.2            0.6    0.94                                     0.2                           0.8    0.46                                                                   0.8    0.6                              E. coli Juhl                                                                          0.19 0.19 0.08 0.19 0.15 0.27 0.95   0.43                                     0.24           0.15           1.7    0.55                                     0.15           0.12           1.8    0.46                                                                   2.2    0.39                                                                   0.8                                     __________________________________________________________________________

ADDITIONAL STARTING MATERIALS6-Chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[4,3b]pyridazin-3-on.

To a solution of 6-chloro-2,3-dihydro-s-triazolo[4,3b]pyridazin-3-on [P.Francabilla and F. Lauria, J. Het. Chem. 8, 415 (1971)] (17 g., 0.1mole) in dry DMF (300 ml.) was added potassium tert.-butoxide (0.5 g.,4.5 m.moles) with stirring. Acrylonitrile (6.6 g., 0.12 mole) in dry DMF(10 ml.) was added to the mixture. The mixture was stirred at 100°-110°C. for 24 hours, then poured into water (700 ml.) and extracted withethyl acetate (5 × 400 ml.). The organic extracts were combined, driedover Na₂ SO₄ and evaporated. The residue was crystallized from ethylacetate to give light yellow needles of6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on(2.5 g., 11%). M.p. 166°-168° C.

ir: ν_(max) ^(KBR) 2230, 1720, 1550, 1500 cm⁻¹.

uv: λ _(max) ^(dioxane) 373 nm (ε2000).

nmr: δ_(ppm) ^(DMSO-d).sbsp.6 3.03 (2H, t, J=6.0 Hz, CH₂), 4.21 (2H, t,J=6.0 Hz, CH₂), 7.23 (1H, d, J=10.0 Hz, pyridazine-H), 7.93 (1H, d,J=10.0 Hz, pyridazine-H).

Anal. Calc'd. for C₈ H₆ N₅ OCl: C, 42.97; H, 2.70; N, 31.32; Cl, 15.86.Found: C, 42.73, 42.56; H, 2.57, 2.50; N, 31.36, 31.68; Cl, 15.96;15.81.

2-(2-Carboxyethyl)-6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on.

A solution of6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on(724 mg.) in 6N-HCl (15 ml.) was refluxed for 6 hours. The reactionmixture was extracted with ethyl acetate (10 × 20 ml.). The combinedextracts were washed with saturated aqueous sodium chloride (50 ml.),dried over Na₂ SO₄ and evaporated to give light yellow, solid2-(2-carboxyethyl)-6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on(567 mg., 72%). M.p.>170° C. (sublimation).

ir: ν_(max) ^(KBr) 3400-2400, 1730, 1710, 1540 cm⁻¹.

uv: λ_(max) ^(dioxane) 377 nmm (ε1500).

nmr: δ_(ppm) ^(D).sbsp. 2^(O+NaHCO).sbsp.3 2.70 (2H, t, J=7.0 Hz, CH₂),4.24 (2H, t, J=7.0 Hz, CH₂), 7.17 (1H, d, J=10.0 Hz, pyridazine-H), 7.70(1H, d, J=10.0 Hz, pyridazine-H).

Anal. Calc'd. for C₈ H₇ N₄ O₃ Cl: C, 39.60; H, 2.91; N, 23.09; Cl,14.61. Found: C, 39.62, 39.48; H, 2.97, 2.67; N, 23.05, 22.70; Cl.13.93, 14.12.

2-(2-Carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolo[4,3b]pyridazin-3-on

A mixture of2-(2-carboxyethyl)-6-chloro-2,3-dihydro-s-triazolo[4,3b]pyridazin-3-on(567 mg., 2.34 m.moles) and 70% sodium hydrosulfide dihydrates (924 mg.,7.02 m.mole) in water (10 ml.) was stirred at room temperature for twohours. The reaction mixture was adjusted successively to ph 1 with c.HCl, to pH 10 with NaOH and then to pH 1 with c. HCl. The resultingprecipitate of2-(carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolo[4,3b]pyridazin-3-onwas collected by filtration and washed with water. Yield: 418 mg. (74%).M.p. 174°-176° C.

ir: ν_(max) ^(KBr) 3600-2600, 2440, 1730, 1720 (sh) cm⁻¹.

uv: λ_(max) ^(pH) 7 buffer 262 nm (ε1700), 318 nm (ε6600).

nmr: δ_(ppm) _(DMSO-d).sbsp.6 2.73 (2 H, t, J=7.0 Hz, CH₂), 4.07 (2H, t,J=7.0 Hz, CH₂), 7.30 (1H, d, J=10.0 Hz, pyridazine-H), 7.74 (1H, d,J=10.0 Hz, pyridazine-H).

Anal. Calc'd for C₈ H₈ N₄ O₃ S: C, 40.00; H, 3.36; N, 23.32; S, 13.35.Found: C, 39.08, 39.06; H, 3.12, 3.20; N, 22.65; 22.70; S, 14.23; 14.29.

7-Amino-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylicAcid

A mixture of 7-ACA (405 mg., 1.49 m.moles), the thiol2-(2-carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolo[4,3b]pyridazin-3-on(357 mg., 1.49 m.moles) and NaHCO₃ (375 mg., 4.47 m.moles) in 0.1 Mphosphate buffer (pH 7, 8 ml.) was stirred at 80° C. for 30 minutes. Thereaction mixture was cooled and filtered to remove insolubles. Thefiltrate was adjusted to pH 1- 2 with c. HCl. The resulting precipitate,7-amino-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b[pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylicacid, was collected by filtration and washed with water. Yield: 519 mg.(77%).

ir: ν_(max) ^(KBr) 3600-2200, 1800, 1725, 1620, 1550, 1480 cm⁻¹.

uv: λ_(max) ^(pH) 7 buffer 253 nm (ε 20,000), 298 nm (ε 10,000).

nmr: δ_(ppm) ^(D).sbsp. 2^(O+K).sbsp.2^(CO).sbsp. 3 2.20 (2H, t, J=7.0Hz, CH₂), 3.40 (1H, d, J=17.5 Hz, 2-H), 3.85 (1H, d, J=17.5 Hz, 2-H),4.00-4.50 (4H, m, 3-CH₂ and N--CH₂ --), 5.01 (1H, d, J=4.0 Hz, 6-H),5.40 (1H, D, J=4.0 Hz, 7-H), 6.94 (1H, d, J=10.0 Hz, pyridazine-H), 7.44(1H, d, J=10.0 Hz, pyridazine-H).

Anal. Calc'd. for C₁₆ H₁₆ N₆ O₆ S₂.3/2 H₂ O: C, 40.09; H, 3.99; N,17.52; S, 13.37. Found: C, 40.06, 40.12; H, 3.33, 3.34; N, 16.96; 16.98;S, 13.87, 13.98.

7-ACA refers to 7-aminocephalosporanic acid and DMF todimethylformamide.

EXAMPLE 87-[o-(N-Butoxycarbonyl-N-methylaminomethyl)phenylacetamido]-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylicacid

To a mixture of7-amino-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylicacid (452 mg., 1 m.mole) and triethylamine (0.46 ml., 3.3 m.mole) in 50%aqueous acetonitrile (4 ml.) was added a THF solution (3ml.) of2,4-dinitrophenylo-(N-t-butoxycarbonyl-N-methylaminomethyl)phenylacetate prepared fromo-(N-t-butoxycarbonyl-N-methylaminomethyl)phenylacetic acid (283 mg.,1.1 m.mole), 2,4-dinitrophenol (202 mg., 1.1 m.mole) and DCC (227 mg.,1.1 m.mole). The mixture was stirred at room temperature overnight andconcentrated under reduced pressure to remove the organic solvents. Theaqueous concentrate was washed with ether (3 × 20 ml.), acidified withc.HCl to pH 1 - 2 and extracted with ethyl acetate (5 × 20 ml.). Thecombined extracts were dried with anhydrous Na₂ SO₄ and evaporated todryness. The residue was chromatographed on a column of silica gel (Wakogel, C-200, 10 g.) by eluting with a mixture of MeOH-- CHCl₃ (MeOH: 0 to3%). The combined eluates which contained the desired product wereevaporated to give 359 mg. (50%) of the title compound. M.P. > 150° C.(dec.).

ir: ν_(max) ^(KBr) 3600-2400, 1780, 1720, 1680, 1550, 1490 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 253 nm (ε19800), 298 nm (ε9400).

nmr: δ_(ppm) ^(DMSO-d).sbsp.6 1.37 (9H, s, t-Bu-H), 2.70 (3H, s,N--CH₃), 2.70 (2H, t, J=7.0 Hz, --CH₂ --), 3.2 - 4.5 (10H, m), 5.01 (1H,d, J=5 Hz, 6-H), 5.60 (1H, d-d, J=5 & 8 Hz, the 8 Hz couplingdisappeared by addition of D₂ O, 7-H), 6.93 (1H, d, J=10 Hz,pyridazine-H). 7.58 (1H, d, J=10 Hz, pyridazine-H).

Anal. Calcd. for C₃₁ H₃₅ N₇ O₉ S₂.5/2H₂ O: C, 49.08; H, 5.31; N, 12.92;S, 8.45. Found: C, 49.32; 49.36; H, 4.70, 4.63; N, 12.52, 12.53; S,8.44, 8.43.

BB-S 525;7-[o-(N-Methylaminomethyl)phenylacetamido]-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylicacid

A mixture of trifluoroacetic acid (1 ml.) and the BOC-protectedcephalosporin prepared above (302 mg., 0.42 m.mole) was allowed to standat room temperature for 15 min. and then diluted with ether (10 ml.).The resulting precipitate was collected by filtration and washed withdry ether (2 × 10 ml.) to afford 263 mg. of solid which was dissolved ina mixture of water (6 ml.) and acetonitrile (3 ml.). The stirredsolution was adjusted at pH 4 with 1 N-NaOH (0.36 ml.) and diluted withacetonitrile (100 ml.) to give the precipitate (187 mg.), which wassuspended in water (4 ml.) and adjusted at pH 9 with sodium hydroxide (1N, 0.3 ml.). The solution was treated with a small amount of activecarbon and freeze-dried to leave the monosodium salt of BB-S 525. Yield106 mg. (39%). M.p. > 180° C. (dec.).

ir: ν_(max) ^(KBr) 3600 - 2400, 1770, 1710, 1600, 1490, 1400 cm⁻¹.

uv: λ_(max) ^(pH) 7 Buffer 253 nm (ε 19,800), 298 nm (ε 8800).

nmr: δ_(max) ^(D).sbsp.2^(O) 2.70 (2H, m, --CH₂ --), 2.75 (3H, s,N--CH₃), 4.4 - 3.4 (10H, m), 4.92 (1H, d, J=4.0 Hz, 6-H), 5.55 (1H, d,J=4.0 Hz, 7-H), 6.93 (1H, d, J=9.5 Hz, pyridazine-H), 7.28 (4H, s,Ph-H), 7.40 (1H, d, J=9.5 Hz, pyridazine-H).

Anal. Calcd. for C₂₆ H₂₆ N₇ O₇ S₂ . Na . 3H₂ O: C, 45.28; H, 4.68; N,14.22; S, 9.30. Found: C, 45.34, 44.84; H, 4.01, 3.85; N, 14.14, 14.08;S, 9.76.

    ______________________________________                                         In vitro antibacterial activity of BB-S 525 compared with                    BB-S 479 and cefamandole (determined by Steers' agar dilution                 method on Mueller-Hinton agar plate)                                          ______________________________________                                                          MIC (mcg./ml)                                               ______________________________________                                        Organism      BB-S 525  BB-S 479  Cefamandole                                 ______________________________________                                        S. aureus Smith                                                                             0.4       0.4       0.2                                         S. aureus     0.2       0.2       0.05                                        S. aureus BX-1633                                                                           0.8       0.8       0.2                                         St. faecalis  >100      >100      100                                         E. coli NIHJ  0.2       0.1       0.1                                         E. coli ATCC 8739                                                                           6.3       3.1       6.3                                         E. coli Juhl  0.4       0.2       0.4                                         E. coli BX-1373                                                                             0.4       0.2       0.2                                         E. coli       0.2       0.1       0.1                                         E. coli       0.2       0.05      0.05                                        E. coli       6.3       3.1       1.6                                         Kl. pneumoniae                                                                              1.6       0.8       3.1                                         Kl. pneumoniae                                                                              0.2       0.1       0.2                                         Kl. pneumoniae                                                                              0.2       0.2       0.8                                         Kl. pneumoniae                                                                              0.2       0.2       0.8                                         Pr. vulgaris  0.2       0.2       0.2                                         Pr. vulgaris  3.1       0.8       50                                          Pr. mirabilis 0.4       0.1       0.4                                         Pr. mirabilis 0.2       0.1       0.2                                         Pr. morganii  >100      >100      3.1                                         pr. morganii  0.2       0.2       0.8                                         Pr. rettgeri  0.8       0.8       0.1                                         Ps. aeruginosa                                                                              >100      >100      >100                                        Ps. aeruginosa                                                                              >100      >100      >100                                        Shig. dysenteriae                                                                           0.1       0.1       0.1                                         Shig. flexneri                                                                              12.5      12.5      3.1                                         Shig. sonnei  0.1       0.1       0.2                                         Serr. marcescens                                                                            25        12.5      50                                          Enterob. cloacae                                                                            3.1       3.1       1.6                                         Sal. enteritidis                                                                            0.2       0.1       0.1                                         Sal. typhosa  0.2       0.1       0.1                                         B. anthracis  0.2       0.2       0.05                                        ______________________________________                                    

EXAMPLE 9

Substitution in the procedure of Example 8 for the2-N-t-butoxycarbonyl-N-methylaminomethyl-4-hydroxyphenylacetic acid usedtherein of an equimolar weight of2-N-t-butoxycarbonylaminomethyl-4-hydroxyphenylacetic acid and of2-N-t-butoxycarbonylaminomethyl-4-methoxyphenylacetic acid and of2-N-t-butoxycarbonyl-N-methylaminomethyl-4-methoxyphenylacetic acid,respectively, produces the compounds having the structures ##STR53##

There is also provided by the present invention a compound having theformula ##STR54## wherein A represents ##STR55## wherein R" is hydrogen,hydroxy or methoxy; R' is hydrogen or methyl; n is one or two and M is##STR56## n is 0 to 4; R is hydrogen, alkyl having 1 to 8 carbon atoms,cycloalkyl of 3 to 6 carbon atoms, phenyl, C₁ -C₄ phenalkyl, pyridyl,thienyl, or pyrrolyl; R¹ is hydrogen, methyl or ethyl; R² and R³ areeach hydrogen, alkyl having 1 to 6 carbon atoms, phenyl, pyridyl, orthienyl; R⁴ and R⁵ are each hydrogen or alkyl of 1 to 4 carbon atoms; R⁶is alkyl having 1 to 4 carbon atoms, phenyl, phenalkyl having 1 to 4carbon atoms, pyridyl, thiadiazolyl, amino or C₁ -C₄ alkylamino; X is NHor oxygen; and each phenyl group is unsubstituted or substituted withone or two substituents selected from the group consisting of alkylhaving 1 to 6 carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy,amino, NHR¹, N(R¹)₂, nitro, fluoro, chloro, bromo or carboxy, or anontoxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a compound having theformula ##STR57## wherein A represents ##STR58## wherein R" is hydrogen,hydroxy or methoxy; R¹ is hydrogen or methyl; n is one or two and M isselected from the group consisting of ##STR59## wherein R⁵ is a hydrogenatom, a methyl or an ethyl group; X² is --O--, --NH--; R⁶ is a basicgroup such as alkyl or aralkyl substituted with substituted orunsubstituted NH₂, such as alkyl-NHCH₃, aralkyl-NHCH₃, ##STR60## R⁷ isan alkyl group such as a methyl, ethyl, propyl, isopropyl, butyl,isobutyl, pentyl or 2-ethyl-hexyl group; a cycloalkyl group such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; an arylgroup such as phenyl or naphthyl; an aralkyl group such as benzyl ornaphthylmethyl; a heterocyclic group and wherein the alkyl, cycloalkyl,aryl, aralkyl and heterocyclic groups may be substituted with one ormore groups selected from the class consisting of amino groups,substituted amino groups such as methylamino, diethylamino or acetamidogroups, the halogen groups such as fluorine, chlorine or bromine, nitrogroups, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy,butoxy or isobutoxy; or a nontoxic, pharmaceutically acceptable saltthereof.

There is also provided by the present invention a compound having theformula ##STR61## wherein A represents ##STR62## wherein R" is hydrogen,hydroxy or methoxy; R' is hydrogen or methyl; n is one or two and M is##STR63## wherein Y is alkyl of one to six carbon atoms, phenyl, benzyl,alkoxy of one to six carbon atoms, or benzyloxy; Z is alkyl of one tosix carbon atoms, phenylbenzyl, alkoxy of one to six carbon atoms,cyclopentyl, cyclohexyl and phenyl, or Y+Z taken together are a3-benzoxazolidine ring; or a nontoxic, pharmaceutically acceptable saltthereof.

Also included within the present invention are pharmaceuticalcompositions comprising a mixture of an antibacterially effective amountof a compound of the present invention and a semisynthetic penicillin oranother cephalosporin or a cephamycin or a β-lactamase inhibitor or anaminoglycoside antibiotic.

There is further provided by the present invention a pharmaceuticalcomposition comprising an antibacterially effective amount of a compoundhaving the formula ##STR64## wherein A represents ##STR65## wherein R"is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; n is one ortwo and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl,acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or5-indanyl and preferably is hydrogen or a nontoxic, pharmaceuticallyacceptable salt thereof.

There is further provided by the present invention a method of treatingbacterial infections comprising administering by injection to aninfected warm-blooded animal, including man, an effective but nontoxicdose of 250-1000 mgm. of a compound having the formula ##STR66## whereinA represents wherein R" is hydrogen, hydroxy or methoxy; R' is hydrogenor methyl; n is one or two and M is hydrogen, pivaloyloxymethyl,acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl,β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl or a nontoxic,pharmaceutically acceptable salt thereof.

There is also provided by the present invention a method for combattingShig. dysenteriae infections which comprises administering to awarm-blooded mammal infected with a Shig. dysenteriae infection anamount effective for treating said Shig. dysenteriae infection of acomposition comprising a compound having the formula ##STR67## wherein Arepresents ##STR68## wherein R" is hydrogen, hydroxy or methoxy; R' ishydrogen or methyl; n is one or two and M is hydrogen,pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl,p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl andpreferably is hydrogen or a nontoxic, pharmaceutically acceptable saltthereof.

There is also provided by the present invention a method for combattingB. anthracis infections which comprises administering to a warm-bloodedmammal infected with a B. anthracis infection an amount effective fortreating said B. anthracis infection of a composition comprising acompound having the formula ##STR69## wherein A represents ##STR70##wherein R" is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; nis one or two and M is hydrogen, pivaloyloxymethyl, acetoxymethyl,methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl,3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic,pharmaceutically acceptable salt thereof.

We claim:
 1. A compound having the formula ##STR71## wherein n is one ortwo and A represents ##STR72## wherein R is hydrogen, hydroxy ormethoxy; R' is hydrogen or methyl; and R² is hydrogen,pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl,p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl.
 2. Anontoxic pharmaceutically acceptable salt of a compound of claim
 1. 3. Acompound having the formula ##STR73## wherein n is one or two and Arepresents ##STR74## wherein R is hydrogen, hydroxy or methoxy or##STR75## and R' is hydrogen or methyl.
 4. A nontoxic pharmaceuticallyacceptable salt of a compound of claim
 3. 5. A compound having theformula ##STR76## wherein n is one or two and R' is hydrogen or methyl.6. A nontoxic, pharmaceutically acceptable salt of a compound of claim5.
 7. A compound of claim 5 having the formula ##STR77## wherein n isone or two.
 8. A nontoxic, pharmaceutically acceptable salt of thecompound of claim
 7. 9. A compound of claim 5 having the formula##STR78## wherein n is one or two.
 10. A compound of claim 1 having theformula ##STR79## wherein n is one or two.
 11. A compound of claim 1having the formula ##STR80## wherein n is one or two.
 12. A compound ofclaim 1 having the formula ##STR81## wherein n is one or two.
 13. Acompound of claim 1 having the formula ##STR82## wherein n is one ortwo.
 14. A compound having the formula ##STR83## wherein A represents##STR84## wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen ormethyl; and R² is hydrogen, pivaloyloxymethyl, acetoxymethyl,methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl,3-phthalidyl or 5-indanyl.
 15. A nontoxic pharmaceutically acceptablesalt of a compound of claim
 14. 16. A compound having the formula##STR85## wherein A represents ##STR86## wherein R is hydrogen, hydroxyor methoxy or ##STR87## and R' is hydrogen or methyl.
 17. A compoundhaving the formula ##STR88## wherein R' is hydrogen or methyl.
 18. Thecompound of claim 17 having the formula ##STR89##
 19. The compound ofclaim 17 having the formula ##STR90##
 20. The compound of claim 1 havingthe formula ##STR91##
 21. The compound of claim 1 having the formula##STR92##
 22. The compound of claim 1 having the formula ##STR93## 23.The compound of claim 1 having the formula ##STR94##
 24. A compoundhaving the formula ##STR95## wherein A represents ##STR96## wherein R ishydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and R² ishydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or5-indanyl.
 25. A nontoxic pharmaceutically acceptable salt of a compoundof claim
 24. 26. A compound having the formula ##STR97## wherein Arepresents ##STR98## wherein R is hydrogen, hydroxy or methoxy or##STR99## and R' is hydrogen or methyl.
 27. A compound having theformula ##STR100## wherein R' is hydrogen or methyl.
 28. The compound ofclaim 27 having the formula ##STR101##
 29. The compound of claim 27having the formula ##STR102##
 30. The compound of claim 1 having theformula ##STR103##
 31. The compound of claim 1 having the formula##STR104##
 32. The compound of claim 1 having the formula ##STR105## 33.The compound of claim 1 having the formula ##STR106##
 34. A compound ofclaim 3 having the formula ##STR107## wherein n is one or two.
 35. Thecompound of claim 16 having the formula ##STR108##
 36. The compound ofclaim 26 having the formula ##STR109##